Abstract:

pH-Responsive polymer-based protein delivery carriers and compositions,
methods for making the carriers and compositions, and methods for using
the carriers and compositions for intracellular protein antigen delivery,
inducing a cytotoxic T-lymphocyte response, introducing a tumor-specific
protein antigen to an antigen presenting cell to induce an immune
response, and providing tumor protection to a subject.

Claims:

2. The conjugate of claim 1, wherein the pH-responsive polymer is a
membrane destabilizing polymer or membrane disrupting polymer.

3. The conjugate of claim 1, wherein the pH-responsive polymer comprises
plurality of repeating units comprising a C2-C8 alkyl group and
a carboxylic acid group ionized at pH 7.4 and protonated at pH 5.5-6.0.

4. The conjugate of claim 1, wherein the pH-responsive polymer comprises a
repeating unit having the formula: ##STR00002## wherein * designates the
point of attachment of the repeat unit to other repeat units and R is a
C2-C8 alkyl group.

5. The conjugate of claim 1, wherein the pH-responsive polymer is a
random, block, or graft copolymer.

6. The conjugate of claim 1, wherein the immunotherapeutic agent is a
protein or peptide therapeutic agent.

7. The conjugate of claim 1, wherein the immunotherapeutic agent is a
protein or peptide antigen.

8. The conjugate of claim 1, wherein the immunotherapeutic agent is a
protein or peptide cancer antigen.

9. The conjugate of claim 1, wherein the immunotherapeutic agent is a
protein or peptide vaccine.

10. A particle, comprising the conjugate of claim 1 and a cationic
complexing agent.

11. A pharmaceutical composition, comprising a pharmaceutically acceptable
excipient and a conjugate of claim 1.

12. A method for delivering a protein or peptide antigen to cell's
cytosol, comprising contacting a cell with a conjugate of claim 1.

13. A method for inducing a cytotoxic T-lymphocyte response, comprising
contacting a cell with the conjugate of claim 1.

14. A method for providing tumor protection to a subject, comprising
administering to a subject a therapeutically effective amount of the
conjugate of claim 1, wherein the immunotherapeutic agent is a protein or
peptide cancer antigen.

15. A method for introducing a tumor-specific protein antigen to an
antigen presenting cell to induce an immune response again the antigen
and cells presenting the antigen, comprising contacting an antigen
presenting cell with the conjugate of claim 1.

16. The method of claim 15, wherein the antigen presenting cells are
selected from dendritic cells, macrophages, and B cells.

[0003]There remains considerable opportunity for the improvement of cancer
treatment strategies, and cancer vaccination, or immunotherapy, is a
promising technique which employs the body's own immune system to
identify and destroy diseased cells. This strategy provides the potential
for increased efficacy over conventional methods, and if used in
combination therapy could reduce the dose of toxic chemotherapeutic
agents and radiation measures that are often accompanied by devastating
side effects. Immunotherapy is especially promising in the prevention of
the metastatic spreading of cancerous cells, which remains a significant
threat even after the successful removal of primary tumor tissue by
standard methods. Improving the efficacy and applicability of
immunotherapy techniques could therefore lead to improved prognosis and
quality of life for cancer patients undergoing treatment.

[0004]Suitable antigens have been identified for vaccination strategies,
but effective delivery of these antigens to elicit a potent immune
response remains a challenge. While substantial research has been
performed in this area, an optimal carrier has yet to be developed. One
considerable challenge is the need for efficient delivery of antigens
into the Class I antigen presentation pathway, accessed primarily in the
cytosol, of antigen presenting cells. This pathway leads to activation of
cytotoxic T-lymphocytes (CTLs), which are capable of direct lysis of
diseased cells and are critical to an effective immunotherapeutic
response. The proposed research aims to increase the efficacy of
therapeutic vaccination by developing a pH-sensitive carrier designed to
increase the cytosolic delivery of antigens, enhancing the CTL immune
response.

[0005]The traditional concept of a vaccine refers to immunization against
bacterial or viral antigens prior to exposure to the actual pathogenic
organism. Most conventional vaccines provide protection primarily through
an antibody-mediated humoral immune response, and to some degree by
stimulation of T-helper cells. The fundamentals of vaccine strategy have
since been applied to cancer treatment. While a humoral immune response
has been associated with a favorable prognosis in some cancers, research
has mainly been focused on enhancing the cellular (T-cell-mediated)
immune response. The activation of CTLs is a particular objective, as
they are the immune component primarily responsible for eliminating tumor
cells, as well as cells infected by some viruses. Unlike the prophylactic
nature of traditional vaccines, cancer vaccination may be most valuable
clinically as a therapeutic measure against previously established
tumors. However, uses as a preventative technique may be applicable in
cases of early identification of pre-cancerous lesions, for individuals
with an increased genetic risk of certain cancers, or in preventing the
metastatic spreading of a previously removed tumor.

[0006]Many tumors are characterized by the over-expression of
tumor-specific protein antigens, and the goal of cancer vaccination is to
identify these antigens and deliver them to the appropriate antigen
presenting cells (APCs) in such a way as to induce an immune response
against the antigen and any cancerous cells expressing it. Professional
APCs possess the co-stimulatory molecules necessary to activate the T
cells they interact with, resulting in an immune response against the
antigen, rather than immune tolerance.

[0007]The professional antigen presenting cells of the immune system
include dendritic cells, macrophages, and B cells. These cells process
and present protein antigens either generated inside the cell (e.g. due
to viral infection) or brought into the cell by sampling of the
extracellular environment. B cells primarily internalize antigen that is
bound to their IgG surface receptors. Macrophages are extraordinarily
efficient at endocytosing antigens, including phagocytosis of large
particulate substances. They present antigens on both MHC1 and MHC2
molecules, but are not as active at antigen presentation as dendritic
cells. Dendritic cells (DCs) are derived from bone marrow and reside
primarily in the peripheral tissues in their immature state, in which
they are highly phagocytic. Dendritic cells are able to effectively
internalize extracellular substances via phagocytosis (a
receptor-dependent uptake of particulate substances), and
macropinocytosis (the engulfing of surrounding extracellular fluid). DCs
are stimulated to undergo maturation by inflammatory or pathogenic
substances including bacterial lipopolysaccharides (LPS), heat shock
proteins, and cytokines such as IL-1, GM-CSF, and TNFα; and such
substances, which also stimulate macrophages, can be co-delivered as
adjuvants in immunotherapy treatments. Mature DCs express a high number
of MHC I and II molecules, as well as T-cell co-stimulatory molecules,
and are able to migrate to secondary lymphoid organs such as the spleen
and lymph nodes where they can interact with T-cells. Their ability to
efficiently process and display antigens for T-cell recognition makes
them a desirable in vivo target for cancer immunotherapy.

[0008]There are two main pathways by which antigens are processed and
presented by APCs for recognition by T-cells. These pathways are depicted
in FIG. 1. The class I pathway results in presentation of antigen
fragments by major histocompatibility complex 1 (MHC1) molecules (in
humans, the corresponding MHC molecules are the human leukocyte antigen,
or HLA, molecules). MHC1 molecules interact with CD8.sup.+ receptors,
leading to activation of cytotoxic T-lymphocytes (CTLs). CTLs are able to
directly lyse diseased cells and play a critical role in effective
immunotherapy. The MHC1 pathway is primarily entered via the cytoplasm of
the APC. Proteins are degraded in the proteasome then further trimmed by
cytosolic peptidases to generate MHC1 peptide epitopes. These peptides
enter the endoplasmic reticulum (ER) via the TAP (transporter associated
with antigen processing) transporter and combine with MHC 1 molecules.
After folding of the MHC1-peptide complex is complete, it is transported
to the cell surface and displayed to naive T-cells.

[0009]The class II presentation pathway is the main pathway by which
exogenous antigens are displayed. These antigens are presented on MHC2
molecules, which can bind to and activate CD4.sup.+ helper T-lymphocytes.
Activated helper T-cells release cytokines that enhance the immune
response and are important to maintaining CTL activity. The combination
of MHC2-restricted peptide epitopes with MHC2 molecules occurs within the
endocytic compartment of the cell.

[0010]Both macrophages, and, to a greater extent, DCs have been shown to
exhibit some degree of cross-presentation, a mechanism by which
extracellular antigens are presented by MHC1 molecules. The exact details
of this process have yet to be determined, but several possible
mechanisms are under investigation including: (1) a direct transport
pathway from the endosome to the cytosol; (2) the delivery of a fraction
of internalized antigen to the ER, where it can then be transported to
the cytosol via TAP; (3) loading of a subset of MHC1 molecules within the
endocytic pathway; and (4) the involvement of a portion of the ER
membrane in the process of phagocytosis. Cross-presentation has been
shown to be most efficient in the case of particulate substances taken up
by the process of phagocytosis. While cross-presentation can occur in
some instances, there remains a considerable opportunity for increasing
the efficiency of antigen delivery to the cytosol in CTL-based
immunotherapeutic strategies. While stimulation of both CD4.sup.+ and
CD8.sup.+ T-cells is required to generate an effective immune response,
activation of CTLs leads to potent antitumor activity and presents a
greater challenge to immunotherapy.

[0011]Antigens can be delivered as whole proteins, peptide antigenic
epitopes, or mRNA or DNA encoding a protein antigen. Delivery of DNA/mRNA
provides the opportunity for an increased supply of antigen since it is
translated multiple times upon reaching the nucleus/cytoplasm. Some
disadvantages to these systems include the added requirement of DNA
delivery into the nucleus, and the high susceptibility of mRNA to
nuclease degradation, as well as the challenge its charge presents to
delivery carrier formulation. The present research focuses on developing
techniques for delivering both proteins and peptides, as the polymer
carrier can be adapted to accommodate both. Delivery of full proteins is
desirable because they are broken down in the proteasome to produce a
range of possible MHC1 and MHC2 antigenic epitopes. This is useful
because, due to HLA genetic variability, not all individuals will possess
an equivalent set of HLA molecules. Delivery of whole protein antigens
also allows the opportunity to activate a greater number of
antigen-specific T-cells. However, obtaining full protein antigens in
large enough amounts for therapeutic treatments can be expensive and time
consuming. Much work has been done in determining the actual peptide
epitopes which are displayed by the MHC molecules. The main limitation of
this strategy is that it does not include all the variations of the
antigen, limiting its usefulness across all members of the population and
decreasing its chances of immune recognition. However, delivery of
peptides is attractive because they can be synthesized chemically, making
them considerably more accessible than full proteins. Furthermore, the
sequences can be enhanced so that they better fit into the MHC complex,
leading to increased T-cell activation.

[0012]Several approaches have been taken toward increasing antigen
delivery in immunotherapy procedures, but there is much potential for
improvement. One method involves removing dendritic cells from the
patient and pulsing them with antigen, then re-injecting them. However,
disadvantages include its high cost and the logistical complications
stemming from the isolation and ex-vivo manipulation of the dendritic
cells. Direct in vivo injection of antigen is advantageous due to its
reduced complexity, lower cost, and decreased invasiveness. Since
delivery of antigen alone has proven largely insufficient in inducing an
immune response, a range of carriers are under development to enhance the
delivery and uptake of injected antigens. Recombinant viral vectors,
which take advantage of the efficient cellular transfection mechanisms of
viruses, have been employed to deliver a variety of antigens, and
conjugation to various bacterial toxins have been employed for similar
reasons. Lipid-based carriers have also been explored for vaccine
delivery. However, certain undesirable qualities of these techniques,
such as safety concerns associated with viral vectors and toxicity and
instability often observed with liposomal systems, has led to the
exploration of synthetic polymer carriers.

[0013]Particles formed from poly(D,L-lactic-co-glycolic acid) (PLGA) have
been widely investigated for antigen delivery and have resulted in
increased MHC1 presentation of encapsulated antigens, a result of more
efficient cross-presentation observed for particulate substances taken up
by phagocytosis compared to soluble substances, as well as the adjuvant
effect observed for PLGA particles. While some endosomal escape has been
observed for this system, a mechanism for cytosolic delivery is not well
established. PLG particles modified to have a cationic surface have also
been explored for the delivery of DNA vaccines. Additionally,
nanoparticulate carriers based on poly(y-glutamic
acid)-poly(L-phenylalanine ethyl ester) have also been developed, and
small 25 nm polypropylene sulfide nanoparticles have been formulated to
specifically target dendritic cells in lymph nodes. One approach that has
been employed to enhance the CTL response to delivered vaccines is the
incorporation of MHC1 adjuvants, such as CpGDNA or TLR ligands, into
carrier formulations. A few groups have investigated an approach similar
to the one presented in this thesis, utilizing pH-responsive carriers to
enhance the CTL response. One such study uses microparticles composed of
dipalmitoylphosphatidylcholine and polymethacrylate Eudragit E100 to
deliver antigenic peptides, and another system utilizes acid degradable
particles and microgels for protein antigen delivery.

[0014]A significant opportunity for improving vaccine antigen delivery
exists in the enhancement of endosomal escape, especially in the case of
soluble, non-particulate antigen complexes. Exogenous substances entering
cells by endocytosis are usually degraded, sequestered, or exocytosed in
the endosomal-lysosomal pathway without gaining access to the cytoplasm.
Enhancing the escape of therapeutic protein antigens from endosomal
compartments could significantly increase the efficacy of protein
vaccines through increased activation of CTLs. This approach is depicted
schematically in FIG. 2.

[0015]Several groups have explored the use of pH-sensitive polymer-based
delivery systems that take advantage of the decreased pH in the endosome.
A popular approach to induce pH-responsive endosomal release has been the
use of systems based on cationic polymers, which enhance endosomal
release via the "proton sponge effect." According to this mechanism, the
polymers become protonated at endosomal pH which leads to an increased
flux of ions into the endosome, causing an increase is osmotic pressure
and eventual disruption of the endosome. However, the high toxicity
associated with the delivery of polycationic substances presents a
considerable challenge to these systems.

[0016]In an effort to enhance endosomal escape and reduce cytotoxicity, a
variety of other polymers with membrane-active capabilities have been
investigated. Carrier particles containing acid-degradable linkages, such
as acetal, ketal, or hydrazone bonds have been developed to degrade more
rapidly in the acidic endosomal environment to trigger release of their
cargo. Several of these systems are being explored for use in antigen
delivery. Block copolymers based on
PEG-poly[(N''-citraconyl-2-aminoethyl)aspartamide] have been used to form
nanocarriers which degrade at endosomal pH and release their cargo due to
repulsive electrostatic force. Block ionomer complexes composed of
graft-comb copolymers of Pluronic (PEO-PPO-PEO) and poly(acrylic acid)
(Pluronic-PolyAA), and a model cationic surfactant,
hexadecyltrimethylammonium bromide, have demonstrated an increase in
positive charge at endosomal pH, which could potentially result in
endosomal membrane interaction. Additionally, polymeric micelles based on
poly(L-lactic acid), PEG, and poly(L-Histidine) and on PEG-poly(L-cystine
bisamide-g-sulfadiazine) have been developed to target the lowered pH
environment associated with tumors for the delivery of hydrophobic
anticancer drugs.

[0017]Despite the advances noted above, a need exists for protein antigen
delivery compositions and methods. The present invention seeks to fulfill
this need and provides further related advantages.

SUMMARY OF THE INVENTION

[0018]The present invention provides protein delivery carriers and
compositions, methods for making the carriers and compositions, and
methods for using the carriers and compositions for intracellular protein
delivery.

[0019]In one aspect, the invention provides a polymer conjugate,
comprising a pH-responsive polymer and one or more therapeutic agents
covalently coupled thereto.

[0020]The pH-responsive polymer is a membrane destabilizing polymer or
membrane disrupting polymer. In one embodiment, the pH-responsive polymer
comprises a plurality of repeating units comprising a C2-C8 alkyl group
and a carboxylic acid group ionized at pH 7.4 and protonated at pH
5.5-6.0.

[0021]The therapeutic agent is a protein or peptide therapeutic agent. In
one embodiment, the therapeutic agent is an immunotherapeutic agent, such
as a protein or peptide antigen. In one embodiment, the immunotherapeutic
agent is a protein or peptide cancer antigen. In one embodiment, the
immunotherapeutic agent is a protein or peptide human cancer antigen. In
one embodiment, the immunotherapeutic agent is a protein or peptide
vaccine.

[0022]In another aspect, the invention provides a particle comprising the
conjugate of the invention and a cationic complexing agent.

[0023]In a further aspect, the invention provides pharmaceutical
compositions. The pharmaceutical composition comprises a pharmaceutically
acceptable carrier and a conjugate or particle of the invention.

[0024]In another aspect, the invention provides a method for delivering a
protein or peptide antigen to cell's cytosol, comprising contacting a
cell with a conjugate or a particle of the invention.

[0025]In another aspect, the invention provides a method for inducing a
cytotoxic T-lymphocyte response, comprising contacting a cell with a
conjugate or a particle of the invention.

[0026]In another aspect, the invention provides a method for providing
tumor protection to a subject, comprising administering to a subject a
therapeutically effective amount of the conjugate of a conjugate or a
particle of the invention, wherein the immunotherapeutic agent is a
protein or peptide cancer antigen.

[0027]In another aspect, the invention provides a method for introducing a
tumor-specific protein antigen to an antigen presenting cell to induce an
immune response against the antigen and cells presenting the antigen,
comprising contacting an antigen presenting cell with a conjugate or a
particle of the invention.

DESCRIPTION OF THE DRAWINGS

[0028]The foregoing aspects and many of the attendant advantages of this
invention will become more readily appreciated as the same become better
understood by reference to the following detailed description, when taken
in conjunction with the accompanying drawings.

[0029]FIG. 1. Summary of MHC-1 and MHC2 pathways. The MHC-1 pathway leads
to activation of CD8+ cytotoxic T-lymphocytes and typically presents
antigens that are processed in the cytoplasm. The MHC2 pathway leads to
activation of CD4+ helper T-lymphocytes and typically presents
exogenous antigens that are internalized into endosomes.

[0031]FIG. 3. Structure of the PPAA-PDSA polymer. Poly(propyl acrylic
acid) (PPAA) is the primary carrier component and becomes increasingly
protonated as the pH decreases, converting from anionic and hydrophilic
at physiological pH to protonated and hydrophobic at the lower pH
observed in the endosome. This enables PPAA to become membrane
interactive at endosomal pH. Pyridyldisulfide acrylate (PDSA), is
incorporated into the polymer at 3-5 mol %, and provides a conjugation
site via disulfide exchange reaction.

[0032]FIG. 4. Schematic of PPAA-PDSA-Ovalbumin conjugation chemistry for
conjugation of ovalbumin to the PDSA moiety of the PPAA-PDSA polymer. The
protein is first modified using Traut's reagent to convert primary amines
to thiols, then the polymer is added and a disulfide exchange reaction
occurs, releasing pyridine-2-thione. Reaction progress can be monitored
by measuring A343 of released pyridine-2-thione.

[0033]FIG. 5. Glutathione reversibility of the polymer-protein
conjugation. In the absence of the cytosolic reducing agent glutathione
(A), little free ovalbumin is present. However, in the presence of 10 mM
glutathione (B), the ovalbumin band reappears due to reduction of the
disulfide bond attaching it to the polymer.

[0034]FIG. 6. pH-dependent hemolysis properties of PPAA polymers and
ovalbumin conjugates. Red blood cells were isolated and added to polymer
and conjugate solutions (polymer concentrations=5 μg/ml) in 0.1M
phosphate buffer. Hemolysis is reported as a percentage of complete lysis
by Triton X-100. PPAA-PDSA shows high membrane-disruptive activity at pH
5.8 but considerably lower membrane disruption at pH 7.4. The polymer
retains its hemolysis capabilities after conjugation to the hydrophilic
protein ovalbumin. PMAA-PDSA, however, does not lyse red blood cells at
any pH due to the decreased hydrophobicity of its methyl side chain
compared with the propyl side chain of PPAA.

[0035]FIG. 7A. CTL activation/MHC-1 presentation of PPAA-ovalbumin
conjugates. Samples were incubated with RAW macrophages for 6 hrs then
removed and B3Z T-cells were added for 16 hrs. Cells were rinsed and
incubated 4 hrs with lysis buffer containing chlorophenol red
β-D-galactoside, then absorbance of released chlorophenol red was
measured at 595 nm. Samples were evaluated in triplicate and errors are
reported as +/-one standard deviation. For reference, the maximum
possible β-galactosidase production was determined by chemically
stimulating the B3Z cells using PMA/ionomycin for 4 hrs, which gave an
A595 of 0.56. Ovalbumin concentration=100 μg/ml. The
PPAA-ovalbumin conjugate shows significantly greater CTL activation than
do any of the control samples (p<0.0005). This is likely due to the
endosomal disruption provided by PPAA, which allows the protein to more
efficiently access the MHC-1 pathway in the cytoplasm. However, PMAA
conjugation results in low CTL activation, similar to that for free
ovalbumin or a PPAA ovalbumin physical mixture (p=0.2). This suggests
that the increase in MHC-1 presentation provided by PPAA is due to its
endosomal disruptive properties rather than solely to increased uptake
due to its larger size compared to free ovalbumin.

[0036]FIG. 7B. Dose-Dependent CTL activation induced by PPAA-ovalbumin
conjugates. Samples were incubated with RAW macrophages at 3
concentrations prior to evaluation with the B3Z CT1s. Samples were
evaluated in triplicate and errors are reported as +/-one standard
deviation. The PPAA-ovalbumin conjugates enhance CTL stimulation in a
dose-dependent manner. It can also be seen that when the polymer ratio is
increased from 1.7 μg/μg ova to 3.2 μg/μg ova, maximal CTL
activation is reached at a lower ovalbumin concentration.

[0037]FIG. 8. Cytotoxicity of PPAA and PMAA polymers and conjugates.
Cytotoxicity was determined for both RAW and B3Z cells using the LDH
assay. Samples were added to cells for 24 hrs at a concentration of 300
μg/ml polymer, twice the highest concentration used in the MHC-1
presentation assay. The cell supernatant was then combined with LDH
reagent and the absorbance at 490 nm was recorded. Percent
survival=1-[(A490 of sample-A490 of untreated cells
control)/(A490 of TritonX control-A490 of untreated cells
control)]×100%. Samples were evaluated in triplicate and error is
expressed as +/-SEM. It can be seen that excessive toxicity was not
observed for any of the samples.

[0038]FIG. 9. Uptake of 14C-Ovalbumin-PPAA by macrophages increases
over time. RAW macrophages were incubated with samples at a concentration
of 50 μg/ml ovalbumin. Cells were washed with PBS and lysed using 1%
Triton X-100. Radioactivity in the cell media, PBS wash, and cell lysate
was measured, and uptake of 14C-ovalbumin was calculated as the %
radioactivity present in the cell lysate compared to the total
radioactivity delivered. Experiments were performed in triplicate and
error is expressed as +/-one standard deviation. It can be seen that
PPAA-conjugated ovalbumin continually accumulates in the cell, whereas
the control sample levels remain fairly constant (p>0.09). This is
likely due to the ability of PPAA-ovalbumin to escape the endosome before
being exocytosed.

[0039]FIG. 10A. Exocytosis study: Total amount of 14C-ovalbumin
internalized during the 1-min uptake time. Samples were incubated with
RAW macrophages for 1 min, then un-internalized conjugate was washed off.
The exocytosis of 14C-ovalbumin into fresh supernatant was followed
for 4 hrs, followed by cell lysis. The amount of ovalbumin internalized
after a 1-min incubation time was determined from the combined exocytosed
and cell-associated radioactivity, as a percentage of the total
delivered. Samples were evaluated with a minimum of n=3 and error is
reported as +/-SEM. It can be seen that the amount of ovalbumin taken
into the cells in 1 min is similar for all samples, giving a uniform
starting point for the exocytosis measurements.

[0040]FIG. 10B. Exocytosis study: Exocytosis profiles following 1-min
uptake time. Following the 1-min incubation of samples with RAW
macrophages, un-internalized conjugate was washed off and the
reappearance of 14C-ovalbumin into fresh supernatant was measured at
various time intervals. After 4 hrs, cells were lysed with 1% Triton
X-100 and the radioactivity in the lysate was measured. The amount of
ovalbumin exocytosed at each timepoint was calculated as a percentage of
the amount initially internalized. Samples were evaluated with a minimum
of n=3 and error is reported as +/-SEM. It can be seen that most
exocytosis occurred in the first 30 minutes, and exocytosis rates were
similar for all samples. However, much less PPAA-conjugated ovalbumin was
exocytosed at each timepoint, compared to controls.

[0041]FIG. 10C. Exocytosis study: Amount of initially internalized
14C-ovalbumin remaining in the cells after 4 hrs exocytosis.
Following the 1-min incubation of samples with RAW macrophages,
un-internalized conjugate was washed off and the reappearance of
14C-ovalbumin into fresh supernatant was measured at various time
intervals. After 4 hrs, cells were lysed with 1% Triton X-100 and the
radioactivity in the lysate was measured. The amount of
14C-ovalbumin remaining in the cells after 4 hrs of exocytosis was
calculated as a percentage of the amount initially internalized. Samples
were evaluated with a minimum of n=3 and error is reported as +/-SEM. The
amount of ovalbumin that remained in the cells and was not exocytosed was
greatly enhanced for PPAA conjugates. This effect is likely due to the
ability of the polymer to disrupt the endosomal membrane and deliver the
ovalbumin to the cytosol before exocytosis can occur.

[0042]FIG. 11A. Exocytosis study: Total amount of 14C-ovalbumin
internalized during the 15-min uptake time. This experiment is similar to
that shown in FIG. 9 except samples were incubated with RAW macrophages
for 15 min rather than 1 min. After 15 min, un-internalized conjugate was
removed and the exocytosis of 14C-ovalbumin into fresh supernatant
was followed for 4 hrs, followed by cell lysis. The amount of ovalbumin
internalized after the 15-min incubation time was determined from the
combined exocytosed and cell-associated radioactivity, as a percentage of
the total delivered. Samples were evaluated with a minimum of n=3 and
error is reported as +/-one standard deviation. The preferential
accumulation of PPAA-ovalbumin is observed for this longer incubation
time.

[0043]FIG. 11B. Exocytosis study: Exocytosis profiles following a 15-min
uptake time. Following the 1-min incubation of samples with RAW
macrophages, un-internalized conjugate was washed off and the
reappearance of 14C-ovalbumin into fresh supernatant was measured at
various time intervals. After 4 hrs, cells were lysed with 1% Triton
X-100 and the radioactivity in the lysate was measured. The amount of
ovalbumin exocytosed at each timepoint was calculated as a percentage of
the amount initially internalized. Samples were evaluated with a minimum
of n=3 and error is reported as +/-one standard deviation. Although the
initial amounts were different for each sample at the beginning of the
study, after normalization to these initial amounts it can be seen that
the exocytosis profiles are very similar to those shown in FIG. 9 for the
1-min uptake time.

[0044]FIG. 11C. Exocytosis study: Amount of initially internalized
14C-ovalbumin remaining in the cells after 4 hrs exocytosis.
Following the 15-min incubation of samples with RAW macrophages,
un-internalized conjugate was washed off and the reappearance of
14C-ovalbumin into fresh supernatant was measured at various time
intervals. After 4 hrs, cells were lysed with 1% Triton X-100 and the
radioactivity in the lysate was measured. The amount of
14C-ovalbumin remaining in the cells after 4 hrs of exocytosis was
calculated as a percentage of the amount initially internalized. Samples
were evaluated with a minimum of n=3 and error is reported as +/-one
standard deviation. The percentage of ovalbumin remaining in the cells
was again greatly enhanced for PPAA conjugates after this longer initial
incubation.

[0045]FIG. 12. Schematic of PPAA-Ova/pDMAEMA ionic particle formation. The
structure of the cationic complexing agent DMAEMA is shown in the top
panel. The 10 kD pDMAEMA used in this study is about 50% protonated at pH
7.4. The lower panel shows a schematic of the particle formulation
process.

[0046]FIG. 13. No cytotoxicity is observed for PPAA and PMAA conjugates or
particles. Toxicity was studied in RAW macrophages using the Alamar blue
assay. Samples were incubated for 24 hrs at a concentration of 750
μg/ml polymer, 5× the amount used for the therapeutic studies. %
survival is shown as a percentage of the untreated cells.

[0047]FIG. 14. Uptake of 14C-Ova is greater for PPAA particles than
for the soluble conjugate. RAW macrophages were incubated with samples at
a concentration of 50 μg/ml of ovalbumin. The cells were then washed
with PBS and lysed using 1% Triton X-100. Radioactivity in the cell
media, PBS wash, and cell lysate was measured, and uptake of
14C-ovalbumin was calculated as the % radioactivity present in the
cell lysate compared to the total radioactivity delivered. Experiments
were performed in triplicate and error is expressed as +/-one standard
deviation. It can be seen that PPAA-ova particle accumulates with time,
to a greater extent than both the soluble PPAA conjugate and the control
PMAA particle.

[0048]FIG. 15A. Exocytosis Study: Exocytosis profiles of
14C-ovalbumin conjugates and particles after a 15 min uptake time.
Samples were incubated with RAW macrophages for 15 min, then
un-internalized samples were removed and cells were washed 2× with
media. Fresh media was added at various time intervals and the
reappearance of 14C-ovalbumin into the supernatant was measured.
After 4 hrs, cells were lysed with 1% Triton X-100 and the radioactivity
in the lysate was measured. The amount of ovalbumin exocytosed at each
timepoint was calculated as a percentage of the amount initially
internalized. Samples were evaluated with a minimum of n=3 and error is
reported as +/-one standard deviation. This figure and the following show
the increased intracellular accumulation/decreased exocytosis achieved by
PPAA conjugates, and to a greater extent PPAA particles. PMAA particles
also show some retention compared to the PMAA conjugate.

[0049]FIG. 15B. Exocytosis study: Amount of initially internalized
14C-ovalbumin remaining in the cells after 4 hrs exocytosis.
Following the 15-min incubation of samples with RAW macrophages,
un-internalized conjugate was washed off and the reappearance of
14C-ovalbumin into fresh supernatant was measured at various time
intervals. After 4 hrs, cells were lysed with 1% Triton X-100 and the
radioactivity in the lysate was measured. The amount of
14C-ovalbumin remaining in the cells after 4 hrs of exocytosis was
calculated as a percentage of the amount initially internalized. Samples
were evaluated with a minimum of n=3 and error is reported as +/-one
standard deviation. This figure summarizes the increased intracellular
accumulation/decreased exocytosis achieved by PPAA conjugates and to a
greater extent PPAA particles, as well as the intermediate retention
achieved by the PMAA particles.

[0050]FIG. 16. Amount of internalized 14C-ovalbumin after a 15 min
uptake time. The amount of ovalbumin internalized after a 15 min
incubation time was determined as a percentage of the total delivered.
Samples were evaluated with a minimum of n=3 and error is reported as
+/-one standard deviation. The preferential accumulation of
PPAA-ovalbumin is observed for this longer incubation time.

[0051]FIG. 17. CTL activation/MHC-1 presentation of PPAA-ovalbumin
particles. RAW macrophages were incubated with samples at a 100 μg/ml
ovalbumin concentration for 4 hrs then washed, and B3Z T-cells were added
for 16 hrs. Cells were again washed and incubated for 2 hrs with lysis
buffer containing chlorophenol red 13-D-galactoside, then the absorbance
of released chlorophenol red was measured at 595 nm. The background
signal of the untreated cells has been subtracted. Samples were evaluated
in triplicate and errors are reported as +/-one standard deviation. It
can be seen that the PPAA particles result in greatly enhanced CTL
activation compared to the soluble PPAA conjugate and the free ovalbumin.
A high concentration ovalbumin sample (5 mg/ml) was used as a positive
control and gave an A595 value of 0.46.

[0052]FIG. 18. Tumor growth curves for ovalbumin-vaccinated mice. C57B1/6
mice (4 per group) were vaccinated with PBS, ovalbumin, or soluble or
particulate PPAA-ova (100 μg ova per mouse), and 7 days later were
injected subcutaneously with E.G7-OVA tumor cells. Tumor size was
measured until tumor volume exceeded 2000 mm3. Error bars represent
the SEM. The excellent tumor protection provided by the PPAA carrier for
the 4-week duration of the study is evidenced in this plot.

[0053]FIG. 19. Anti-ovalbumin IgG antibody concentration in mouse plasma.
C57B1/6 mice (4 per group) were vaccinated with PBS, ovalbumin, or
soluble or particulate PPAA-ova (100 μg ova per mouse). 20 days after
vaccine injection, blood was drawn and ELISA was performed plasma
samples. Error bars represent the SEM. The anti-ova antibody response
induced by the PPAA soluble and particulate conjugates is much greater
than that resulting from vaccination with free ovalbumin.

[0054]FIG. 20. Ovalbumin-reactive CD8+ splenocytes. C57B1/6 mice (4 per
group) were vaccinated with PBS, ovalbumin, and soluble and particulate
PPAA-ova (100 μg ova per mouse. 7 days later spleens were collected
and splenocytes were stained with FITC-anti-CD8 and PE-MHC-1/SIINFEKL
tetramers. Errors bars represent the SEM. The enhanced anti-ova CTL
response induced by the PPAA soluble and particulate conjugate can be
seen.

[0055]FIG. 21. Dose-Dependent CTL Activation/Class I antigen presentation
by DCs pulsed with SLLMWITQC peptides. Primary DCs were incubated for 4
hours at increasing peptide concentrations. Maturation was then induced
and CTLs were added for another 18 hours. IFN-γ production by
activated CTLs was measured by ELISA. Samples were evaluated in
duplicate. The PPAA-peptide provides dose-dependent CTL activation,
though not to as great an extent as the free peptide.

[0056]FIG. 22. CTL Activation/Class I antigen presentation by DCs pulsed
with CAARSLLMWITQV peptides conjugated to PPAA. Primary DCs were
incubated with samples for 4 hours, then maturation was induced and CTLs
were added for another 18 hours. IFN-γ production by activated CTLs
was measured by ELISA. Samples were evaluated in duplicate, and the
concentrations presented are those of the peptide. The modified peptide
conjugated to PPAA results in some dose-dependent CTL activation, though
not to as great an extent as the native unmodified peptide.

[0057]FIG. 23. CTL Activation/Class I antigen presentation by DCs using
BFA inhibitor. Primary DCs were incubated with samples for 4 hours.
Maturation was then induced and CTLs were added for another 18 hours.
IFN-γ production by activated CTLs was measured by ELISA. Peptide
concentration are 1 ug/ml for all samples except the native SLLMWITQV
peptide, which is 0.1 ug/ml. Samples were evaluated in duplicate. Again,
PPAA-peptide does not result in as much CTL activation as does the free
peptide. Some reduction is seen when the BFA inhibitor is employed, but
it is not substantial and occurs to the same extent for the free peptide
samples as for the conjugate.

DETAILED DESCRIPTION OF THE INVENTION

[0058]The present invention provides protein delivery carriers and
compositions, methods for making the carriers and compositions, and
methods for using the carriers and compositions for intracellular protein
delivery.

[0059]In one aspect, the invention provides a polymer conjugate,
comprising a pH-responsive polymer and one or more therapeutic agents
covalently coupled thereto.

[0060]The pH-responsive polymer is a membrane destabilizing polymer or
membrane disrupting polymer. In one embodiment, the pH-responsive polymer
comprises a plurality of repeating units comprising a C2-C8 alkyl group
and a carboxylic acid group ionized at pH 7.4 and protonated at pH
5.5-6.0. In one embodiment, the pH-responsive polymer comprises a
repeating unit selected from C2-C8 alkyl acrylic acid repeating units. In
one embodiment, the pH-responsive polymer comprises a repeating unit
selected from ethyl acrylic acid and propylacrylic acid repeating units.
In one embodiment, the pH-responsive polymer is a poly(propylacrylic
acid). The pH-responsive polymer can be a random, block, or graft
copolymer.

[0061]As used herein, the term "membrane destabilizing" refers to a
polymer or composition that directly or indirectly elicit a change (e.g.,
a permeability change) in a cellular membrane structure (e.g., an
endosomal membrane) so as to permit an agent (e.g., protein or peptide)
to pass through such membrane structure, for example, to enter a cell or
to exit a cellular vesicle (e.g., an endosome). A membrane destabilizing
polymer can be (but is not necessarily) a membrane disruptive polymer. A
membrane disruptive polymer can directly or indirectly elicit lysis of a
cellular vesicle or otherwise disrupt a cellular membrane (e.g., as
observed for a substantial fraction of a population of cellular
membranes). Generally, membrane destabilizing or membrane disruptive
properties of polymers can be assessed by various means. In one
non-limiting approach, a change in a cellular membrane structure can be
observed by assessment in assays that measure (directly or indirectly)
release of an agent (e.g., protein or peptide) from cellular membranes
(e.g., endosomal membranes), for example, by determining the presence or
absence of such agent, or an activity of such agent, in an environment
external to such membrane. Another non-limiting approach involves
measuring red blood cell lysis (hemolysis), e.g., as a surrogate assay
for a cellular membrane of interest. Such assays may be done at a single
pH value or over a range of pH values.

[0062]The terms "endosome disruptive" and "endosomolytic" refers to a
polymer or composition having the effect of increase the permeability of
the endosomal membrane of an endosome.

[0063]The phrase "pH-responsive, membrane-destabilizing or "pH-dependent,
membrane-destabilizing" refers to a polymer or composition that is at
least partially, predominantly, or substantially hydrophobic and is
membrane destabilizing in a pH dependent manner. In certain instances, a
pH-dependent membrane destabilizing polymer block is a hydrophobic
polymeric segment of a copolymer and/or comprises a plurality of
hydrophobic species; and comprises a plurality of chargeable species. In
some embodiments, the chargeable species is anionic. In some embodiments,
the anionic chargeable species is anionic at about neutral pH. In further
or alternative embodiments, the anionic chargeable species is non-charged
at a lower, e.g., endosomal pH. In some embodiments, the membrane
destabilizing chargeable hydrophobe comprises a plurality of cationic
species. The pH dependent membrane-destabilizing chargeable hydrophobe
comprises a non-peptidic and non-lipidic polymer backbone. For example, a
pH dependent, membrane-destabilizing block may possess anionic repeat
units the substituents of which are predominantly ionized (anions) at one
pH, e.g., pH 7.4, and predominantly neutral at a lesser pH, e.g., pH 5.0
whereby the pH dependent, membrane-destabilizing group or block becomes
increasingly hydrophobic as a function of the drop in pH from 7.4 to 5.0.

[0064]The polymeric carrier described herein is a pH-dependent, membrane
destabilizing carrier. The polymeric carrier can be a random, block, or
graft copolymer.

[0065]The polymeric carrier is or comprises a plurality of monomeric
anionic residues and, optionally one or more cationic, zwitterion, or
neutral monomeric residues. In specific embodiments, the plurality of
anionic monomeric residues is anionic at about neutral pH (e.g.,
physiological pH). In more specific embodiments, at least 50%, at least
70%, at least 80%, at least 90%, at least 95%, or at least 99% of the
plurality of anionic monomeric residues are non-charged at about pH 5, at
about pH 5.5, at about pH 5.7, at about pH 6.0, at about pH 6.2, at about
pH 6.5, or at about endosomal pH (e.g., as calculated from the pKa value
of the given monomeric residue).

[0066]In certain embodiments, the plurality of monomeric residues can be
derived from polymerization of (C2-C8) alkyl ethacrylate, a
(C2-C8) alkyl methacrylate, or a (C2-C8) alkyl
acrylate (each of which may be optionally substituted).

[0067]The polymeric carrier may contain anionic repeat units, cationic
repeat units, zwitterionic repeat units, a combination of two or more
charged repeat units (e.g., anionic and cationic repeat units, anionic
and zwitterionic repeat units, cationic and zwitterionic repeat units, or
anionic, cationic and zwitterionic repeat units), substantially
non-charged repeat units, or a combination thereof, provided that its
overall character is pH-responsive and membrane disruptive. The polymeric
carrier may contain any of a wide range of repeat units, hydrophobic or
even hydrophilic, provided that the sum of the contributions of the
repeat units comprised by the carrier provides a polymer having an
overall pH-responsive character. When the repeat units contain ionizable
groups, the contribution of an individual repeat unit to the overall
hydrophilicity of the block of which it is a constituent may vary as a
function of its pKa relative to the pH of the environment in which it is
found. For example, propyl acrylic acid repeat units,
--CH2C(CH2CH2CH3)(COOH)--, are predominantly ionized
at pH 7 but not at pH 5 and thus, the hydrophobic contribution of propyl
acrylic acid repeat units to a block is significantly greater at pH 5
than at pH 7. In general, therefore, it is preferred that the sum of the
contributions of the repeat units constituting the polymeric carrier be
such that the overall character of the block is hydrophobic at pH's that
are less than physiological pH. For example, in one embodiment, the sum
of the contributions is such that the overall character of the carrier is
hydrophobic at a pH of about 5.0. By way of further example, in one
embodiment, the sum of the contributions is such that the overall
character of the carrier is hydrophobic at a pH of about 5.5. By way of
further example, in one embodiment, the sum of the contributions is such
that the overall character of the carrier is hydrophobic at a pH of about
6.0. By way of further example, in one embodiment, the sum of the
contributions is such that the overall character of the carrier is
hydrophobic at a pH of about 6.8. By way of further example, in one
embodiment, the sum of the contributions of the repeat units is such that
the overall character of the carrier is hydrophobic at a pH within the
range of about 6.2 to 6.8.

[0068]In certain embodiments, the polymeric carrier described herein
comprises monomeric residues resulting from the polymerization or
copolymerization of a monomer comprising a hydrophobic species. Monomers
comprising a hydrophobic species include, by way of non-limiting example,
optionally substituted, (C2-C8)alkyl-ethacrylate, a
(C2-C8)alkyl-methacrylate, a (C2-C8)alkyl-acrylate,
styrene, (C2-C8)alkyl-vinyl, or the like.

[0069]In certain embodiments, the polymeric carrier described herein
comprises a plurality of first monomeric residues derived from a first
polymerizable monomer having a protonatable anionic species and a
hydrophobic species, and optionally a plurality of second monomeric
residues derived from a second polymerizable monomer having a
deprotonatable cation species.

wherein * designates the point of attachment of the repeat unit of formula
(I) to other repeat units; and R is a C2-C8 alkyl group. Representative
C2-C8 alkyl groups include methyl, ethyl, and straight chain and branched
propyl, butyl, pentyl, hexyl, heptyl, and octyl groups. In one
embodiment, R is ethyl. In another embodiment, R is n-propyl.

[0071]In one embodiment, the polymeric carrier is a random copolymer
comprising repeat units corresponding to formula (I). In general, the
polymeric carrier comprises a plurality of repeat units, i.e., at least
two. In certain embodiments, a the polymeric carrier described herein has
a number average molecular weight of about 1,000 Dalton to about 200,000
Dalton, about 1,000 Dalton to about 100,000 Dalton, about 1,000 Dalton to
about 100,000 Dalton, about 5,000 Dalton to about 50,000 Dalton, about
10,000 Dalton to about 50,000 Dalton, about 15,000 Dalton to about 35,000
Dalton, or about 20,000 Dalton to about 30,000 Dalton.

[0072]In another embodiment, the polymeric carrier is a block copolymer
comprising ethylenically unsaturated monomers. The term "ethylenically
unsaturated monomer" is defined herein as a compound having at least one
carbon double or triple bond. Suitable ethylenically unsaturated monomers
include alkyl (alkyl)acrylates, methacrylates, acrylates,
alkylacrylamides, methacrylamides, and acrylamides.

[0074]In some embodiments, polymer carriers have a low polydispersity
index (PDI) or differences in chain length. Polydispersity index (PDI)
can be determined in any suitable manner, e.g., by dividing the weight
average molecular weight of the polymer chains by their number average
molecular weight. The number average molecule weight is sum of individual
chain molecular weights divided by the number of chains. The weight
average molecular weight is proportional to the square of the molecular
weight divided by the number of molecules of that molecular weight.
Because the weight average molecular weight is always greater than the
number average molecular weight, polydispersity is always greater than or
equal to one. As the numbers come closer and closer to being the same,
i.e., as the polydispersity approaches a value of one, the polymer
becomes closer to being monodisperse in which every chain has exactly the
same number of constitutional units. Polydispersity values approaching
one are achievable using radical living polymerization. Methods of
determining polydispersity, such as, but not limited to, size exclusion
chromatography, dynamic light scattering, matrix-assisted laser
desorption/ionization chromatography and electrospray mass chromatography
are well known in the art. In some embodiments, the polymer carriers have
a polydispersity index (PDI) of less than 2.0, or less than 1.8, or less
than 1.6, or less than 1.5, or less than 1.4, or less than 1.3, or less
than 1.2.

[0075]The polymer carriers useful in the invention are
membrane-destabilizing at a pH of about 6.5 or lower, preferably at a pH
ranging from about 5.0 to about 6.5, or at a pH of about 6.2 or lower,
preferably at a pH ranging from about 5.0 to about 6.2, or at a pH of
about 6.0 or lower, preferably at a pH ranging from about 5.0 to about
6.0. For example, in one embodiment, the polymer is
membrane-destabilizing at a pH of or less than about 6.2, of or less than
about 6.5, of or less than about 6.8, of or less than about 7.0. In
certain embodiments, membrane destabilization is of any cellular membrane
such as, for example, an extracellular membrane, an intracellular
membrane, a vesicle, an organelle, an endosome, a liposome, or a red
blood cell. In some embodiments, polymeric carriers are membrane
destabilizing (e.g., in an aqueous medium) at an endosomal pH.

[0076]In some embodiments, the polymeric carrier is hemolytic at pH of or
less than about 6.2, of or less than about 6.5, of or less than about
6.8, of or less than about 7.0. In further or alternative embodiments,
the polymeric carrier is substantially non-hemolytic at pH greater than
about 7.0. In specific embodiments, the polymeric carrier is hemolytic at
given concentration and a pH of about 6.2, and substantially
non-hemolytic at the same concentration and at a pH greater than about
7.0. In certain embodiments, the hemolytic nature of the polymer is
determined in any suitable manner, e.g., by use of any standard hemolysis
assay, such as an in vitro hemolysis assay.

[0077]In certain embodiments, the polymeric carrier is endosome
disruptive. In some embodiments, the polymeric carrier is endosome
disruptive at pH of or less than about 6.2, of or less than about 6.5, of
or less than about 6.8, of or less than about 7.0. The endosome
disruptive nature of the polymeric carrier is determined in any suitable
manner, e.g., by use of any standard hemolysis assay, such as an in vitro
endosomolysis assay, or an in vivo non-human mammal endosomolysis assay.

[0078]The invention provides a polymer conjugate, comprising a
pH-responsive polymer and one or more therapeutic agents covalently
coupled thereto. As used herein, therapeutic agent therapeutic agent
refers to an agent that, when administered to a subject, organ, tissue,
or cell has a therapeutic effect and/or elicits a desired biological
and/or pharmacological effect. In one embodiment, the therapeutic agent
is an immunotherapeutic agent, more specifically, a protein or peptide
therapeutic agent. In one embodiment, the immunotherapeutic agent is a
protein or peptide antigen. In one embodiment, the immunotherapeutic
agent is a protein or peptide cancer antigen. In one embodiment, the
immunotherapeutic agent is a protein or peptide human cancer antigen. In
one embodiment, the immunotherapeutic agent is a protein or peptide
vaccine.

[0079]The immunotherapeutic agent is covalently coupled to the polymer. In
one embodiment, the immunotherapeutic agent is covalently coupled to the
polymer through a disulfide linkage.

[0080]In another aspect, the invention provides a particle comprising the
conjugate of the invention and a cationic complexing agent. Suitable
cationic complexing agents include any cationic agent capable of
associating with a conjugate of the invention to provide a particle
(e.g., ionic particle). Representative cationic complexing agents include
cationic polymers (e.g., pDMAEMA). In contrast to the conjugates of the
invention, which are soluble in aqueous media, the particles of the
invention are non-soluble.

[0081]In a further aspect, the invention provides pharmaceutical
compositions. The pharmaceutical composition comprises a pharmaceutically
acceptable carrier and a conjugate or particle of the invention.

[0082]Formulations comprising the pH-responsive polymer compositions of
the invention (i.e., conjugates and particles) can be pharmaceutical
compositions. Such pharmaceutical compositions can comprise, for example,
a composition of the invention and a pharmaceutically acceptable
excipient.

[0083]In some embodiments, the pH-responsive polymer composition is
administered to a patient in any suitable manner, e.g., with or without
stabilizers, buffers, and the like, to form a pharmaceutical composition.
In some embodiments, the pH-responsive polymer composition is formulated
and used as tablets, capsules or elixirs for oral administration,
suppositories for rectal administration, sterile solutions, suspensions
or solutions for injectable administration, and any other suitable
compositions.

[0084]In some embodiments, pharmaceutical compositions comprising the
pH-responsive polymer composition are administered systemically. As used
herein, "systemic administration" means in vivo systemic absorption or
accumulation of drugs in the blood stream followed by distribution
throughout the entire body. Administration routes which lead to systemic
absorption include, without limitation: intravenous, subcutaneous,
intraperitoneal, inhalation, oral, intrapulmonary and intramuscular. In
some embodiments, the compositions are administered topically.

[0085]In some embodiments, the compositions are prepared for storage or
administration and include a pharmaceutically effective amount of the
pH-responsive polymer composition in a pharmaceutically acceptable
carrier or diluent. Any acceptable carriers or diluents are optionally
utilized herein. Specific carriers and diluents and are described, e.g.,
in Remington's Pharmaceutical Sciences, Mack Publishing Co., A. R.
Gennaro Ed., 1985. As used herein, the term "pharmaceutically acceptable
carrier" means a non-toxic, inert solid, semi-solid or liquid filler,
diluent, encapsulating material or formulation auxiliary of any type. In
some embodiments, the pharmaceutical compositions provided herein are
administered to humans and/or to animals, orally, rectally, parenterally,
intracistemally, intravaginally, intranasally, intraperitoneally,
topically (as by powders, creams, ointments, or drops), bucally, or as an
oral or nasal spray.

[0087]The pH-responsive polymer compositions of the invention are used for
intracellular delivery of a therapeutic agent (i.e., protein or peptide).
The composition can be exposed to and contacted with a cell surface
(e.g., via directed targeting) in a medium at a first pH. The composition
is introduced into an endosomal membrane within the cell, for example,
through endocytosis and, in some embodiments, through receptor mediated
endocytosis. The endosomal membrane is destabilized (e.g., by the
polymeric carrier, which is a membrane destabilizing polymer), thereby
delivering the therapeutic agent (e.g., protein or peptide) to the
cytosol of the cell.

[0088]In one aspect, the invention provides a method for delivering a
protein or peptide antigen to cell's cytosol, comprising contacting a
cell with a conjugate or a particle of the invention.

[0089]In another aspect, the invention provides a method for inducing a
cytotoxic T-lymphocyte response, comprising contacting a cell with a
conjugate or a particle of the invention.

[0090]In another aspect, the invention provides a method for providing
tumor protection to a subject, comprising administering to a subject a
therapeutically effective amount of the conjugate of a conjugate or a
particle of the invention, wherein the immunotherapeutic agent is a
protein or peptide cancer antigen. The term "therapeutically effective
amount" of a therapeutic agent means an amount that is sufficient, when
administered to a subject suffering from or susceptible to a disease,
disorder, and/or condition, to treat, diagnose, prevent, and/or delay the
onset of the symptom(s) of the disease, disorder, and/or condition.

[0091]In another aspect, the invention provides a method for introducing a
tumor-specific protein antigen to an antigen presenting cell to induce an
immune response against the antigen and cells presenting the antigen,
comprising contacting an antigen presenting cell with a conjugate or a
particle of the invention. Representative antigen presenting cells
include dendritic cells, macrophages, and B cells.

[0092]As noted above, in one aspect, the invention provides
immunotherapeutic conjugates that include at least two components: (1) a
polymeric carrier, a synthetic polymer consisting of hydrophobic alkyl
groups and acidic carboxyl groups with polymer compositions chosen so
that the carboxyl groups are ionized at physiological pH and (2) one or
more immunotherapeutic agents covalently coupled thereto.

[0093]The polymers remain inactive in their ionized, hydrophilic state at
pH 7.4. However, at the decreased pH levels typical of the endosome (pH
5.5-6.0), the carboxyl groups become protonated and the polymers
transition to a state that is membrane destabilizing. This mechanism
directly mimics that of viral and other pathogenic proteins, such as
hemaglutinin and diphtheria toxin, that have evolved to enhance endosomal
escape, and of synthetic fusogenic peptides such as GAL4 designed to
imitate this behavior. The mechanistic utility of pathogenic proteins has
demonstrated the importance of endosomal release in MHC1 presentation and
CTL activation, but corresponding biological responses to the pathogenic
proteins has limited their clinical translation.

[0094]The present invention provides pH-responsive, membrane-destabilizing
polymeric carriers for cytosolic protein antigen delivery. A
representative polymeric carrier of the invention is illustrated in FIG.
3. Referring to FIG. 3, this poly(propylacrylic acid-co-pyridyldisulfide
acrylate) (PPAA-PDSA) polymer is designed to conjugate a protein antigen
by disulfide exchange between thiolated protein and the PDSA monomers.
The resulting protein-polymer disulfide bond can be reduced by the
cytosolic glutathione reduction system to leave the antigen free for
subsequent proteolytic processing. The formulation of this carrier and
its ability to enhance MHC1 presentation and CTL activation in protein
immunotherapeutic strategies is described herein.

[0095]The following describes the synthesis of a representative polymer
carrier of the invention (PPAA-PDSA) and its conjugation to a model
protein (ovalbumin); the evaluation of the ability of this conjugate to
enhance MHC1 presentation in macrophages; the uptake and intracellular
accumulation of PPAA conjugates using radiolabeled ovalbumin; a
particulate PPAA-ovalbumin formulation prepared by incorporating a
cationic complexing agent, the uptake and intracellular accumulation of
these particles, and the ability of the particles to increase MHC1
presentation of ovalbumin in a dendritic cell line; in vivo evaluation of
both the soluble and particulate PPAA formulations in an
ovalbumin-expressing tumor model in mice; and a study in which the
ability of PPAA to deliver tumor antigenic MHC1 peptides to primary
dendritic cells.

[0096]Conjugation of Poly(Propylacrylic Acid) Enhances the MHC-1
Presentation of Ovalbumin in a Macrophage Cell Line

[0097]One considerable barrier to effective immunotherapy strategies is
delivery of the therapeutic antigen into the MHC-1 presentation pathway,
accessed primarily in the cytosol, of antigen presenting cells. This
pathway leads to activation of the potent cytotoxic T-lymphocytes, which
are able to destroy diseased cells. This study utilizes a synthetic,
pH-responsive carrier based on poly(propylacrylic acid) (PPAA) to enhance
cytosolic delivery and subsequent MHC-1 presentation of the model protein
antigen ovalbumin. Increased cytosolic delivery is achieved due to the
membrane destabilizing action of PPAA in response to the acidic endosomal
pH following cellular uptake of polymer-antigen conjugates. This system
mimics the endosomal escape mechanism employed by viral and microbial
agents to access the cytoplasm of host cells, while circumventing the
toxicity and immunogenicity associated with these pathogenic agents. The
carrier is designed to incorporate ovalbumin via a disulfide bond, which
can then be reduced by the cytosolic glutathione reduction system to
leave the antigen free for subsequent proteolytic processing.

[0098]Ovalbumin-PPAA conjugates are capable of pH-sensitive membrane
disruption, as determined by a red blood cell hemolysis assay. These
results suggest that the PPAA-antigen conjugates should be capable of
escaping the endosome, allowing ovalbumin to access the MHC-1 pathway.
This was then tested using a cytotoxic T-lymphocyte (CTL) activation
assay. This model utilizes a CD8+ T-cell hybridoma that is activated upon
T-cell receptor ligation to an MHC-1/ovalbumin peptide complex displayed
from a model RAW macrophage cell. The PPAA-ovalbumin conjugates exhibited
considerable increases in the MHC-1 presentation of ovalbumin, compared
to negative controls: free ovalbumin, ovalbumin and PPAA mixed
physically, and ovalbumin conjugated to a non membrane-active polymer,
poly(methacrylic acid). Finally, toxicity of these carriers was evaluated
in both the macrophage and T-cell lines and found to be negligible.
Together, these results suggest PPAA-based delivery systems are
well-suited for enhancing the efficacy of therapeutic protein vaccines.

[0099]The conjugation scheme for attaching ovalbumin to PPAA-PDSA is
described in Example 1 and shown in FIG. 4. The conjugates are formed
through disulfide exchange of the ovalbumin thiol with the PDSA group in
the polymer, and are designed to be reduced by glutathione in the cell
cytoplasm, freeing the protein for processing and MHC-1 presentation.

[0100]The conjugation reaction highly favors polymer-protein combinations
over polymer-polymer and protein-protein combinations. Pyridine-2-thione
is a strong leaving group, making disulfide exchange between the thiol on
the protein and the disulfide bond in PDSA much more likely than
formation of a disulfide bond between two thiols on protein molecules.
The polymer contains very few thiols to initiate disulfide exchange, as
determined by measuring the initial A343 of potentially dissociated
PDSA in the polymer sample prior to conjugation. If such a reaction were
to occur, the resulting polymer-polymer disulfide would likely be
exchanged with a protein due to the higher concentration of protein
thiols.

[0101]The glutathione reversibility of the conjugation is demonstrated in
FIG. 5. This is an important feature as it allows the protein to be
readily freed from the polymer carrier once it reaches the cytoplasm. The
experiment was performed using a physiological glutathione concentration
of 10 mM. However, the amount of glutathione able to reduce the
polymer-protein disulfide bond is likely greater in the cytosol, due to
continued regeneration of reactive glutathione by the enzyme glutathione
reductase.

[0102]Red Blood Cell Hemolysis. Red blood cell hemolysis assays were
performed to evaluate the pH-dependent membrane disruptive activity of
the polymers and polymer-protein conjugates. Lysis of red blood cells
demonstrates the ability of the polymer to disrupt lipid membranes and
has been shown to correlate with endosome disruption. Membrane
destabilization results from the hydrophilic to hydrophobic transition
that occurs near the pKa of the PPAA polymer. At pH 7.4, the carboxyl
groups making up the polymer backbone are present primarily in their
ionized form, making the polymer hydrophilic. As the pH decreases, the
carboxyl groups become protonated, and this resulting increase in
hydrophobicity allows the polymer to destabilize the cell membrane,
releasing hemoglobin.

[0103]Hemolysis assays were conducted at three pH values (7.4, 6.6, and
5.8) in order to approximate physiological conditions as well those
encountered in the early and late endosomes. Conjugate concentrations
were adjusted to contain 5 μg/ml of polymer. The results of this assay
are detailed in FIG. 6. As expected, the PPAA-PDSA polymer and conjugates
were considerably more hemolytic at the lower pH characteristic of the
endosome (pH 5.8) than at physiological pH: the PPAA polymer demonstrated
90% hemolysis at pH 5.8 and only 30% hemolysis at pH 7.4.

[0104]The PPAA-PDSA polymer shows slightly higher hemolysis at
physiological pH than does a PPAA homopolymer, which consistently gave
less than 10% hemolysis at pH 7.4 while exhibiting close to 100%
hemolysis at pH 5.8. This increased hemolysis at pH 7.4 is likely due to
the hydrophobicity added by the PDSA monomer, especially the pyridyl
group. The increased hemolysis at pH 7.4 has not resulted in increased
cytotoxicity at the concentrations and incubation times used, especially
as many of these groups are released during the protein conjugation
reaction. However, should this become a significant concern, excess
pyridyl groups can be released using a thiol containing compound. The
PPAA-ovalbumin conjugates have similar hemolysis profiles to the
PPAA-PDSA polymer alone, indicating that the polymer retains its
hemolytic activity when attached to a 40 kD hydrophilic protein. The
retention of pH-sensitive hemolytic behavior in the conjugates is
important because it indicates that the conjugates will be able to escape
to the cytoplasm following endosomal uptake.

[0105]In contrast to the PPAA polymers, which become protonated at
endosomal pH and are sufficiently hydrophobic to interact with lipid
membranes, the PMAA polymer and conjugate were not hemolytic at any pH.
This is because PMAA does not possess sufficient hydrophobic character to
interact with membranes at any of the pH values tested. Since PMAA is
similar in nature to PPAA but is not hemolytic, it is a potentially
useful negative control polymer with which to compare the antigen
delivery capabilities of PPAA.

[0106]The ability of the polymer to enhance delivery of protein antigen
from the endosome to the cytoplasm of APCs and into the MHC class I
antigen presentation was evaluated using the LacZ class I presentation
assay described in Example 1. The PPAA-PDSA-ovalbumin conjugate resulted
in a strong 22-fold increase in MHC-I presentation and CTL activation
compared to free ovalbumin (p=0.0001), as shown in FIG. 7A.

[0107]It can also be seen in this figure that the ovalbumin must be
chemically attached to the PPAA in order to be effectively delivered into
the MHC-1 pathway, as physical mixtures showed only background CTL
activation levels (p=0.20 compared to free ovalbumin). Conjugation to
PPAA also resulted in much greater CTL activation than did conjugation to
the non membrane-disruptive polymer, PMAA (p=0.0002, 11-fold increase in
CTL activation The PMAA-ovalbumin conjugates did not significantly
enhance CTL activation over delivery of free ovalbumin (p=0.72.) These
results correlate the membrane destabilizing activity of the polymer to
the level of MHC-1 presentation and CTL activation, and suggest that the
increase in presentation is not solely the result of increased cellular
uptake due to the larger size of the conjugate compared to free
ovalbumin.

[0108]Additionally, PPAA conjugation increases the CTL activation in a
dose-dependent manner, whereas higher concentrations of control samples
do not result in significant increases in CTL activation (p<0.2) (FIG.
7B). Increasing the amount of PPAA in the conjugate further exaggerates
this effect: when the weight ratio of polymer:protein is doubled from 1.7
to 3.2 μg polymer per μg protein, CTL activation increases,
becoming maximized at a lower ovalbumin concentration. The overall
increases in CTL activation provided by conjugation to PPAA in comparison
with the various control groups are summarized in Table 1.

[0109]The PPAA carrier performs well when compared to other protein
delivery systems which have been evaluated using the B3Z CTL activation
assay. Acid-degradable particles used to deliver ovalbumin have resulted
in maximal A595 values (the measure of CTL activation) in the range
of 0.25-0.4, which is similar to that reported in our study, and PLGA
particles have been shown to provide a 5-fold increase in CTL activation
over delivery of ovalbumin alone. While direct comparisons are not
possible due to the different carrier architectures (soluble vs.
particulate), and differences in experimental conditions and data
presentation, it is useful to put the PPAA carrier into the context of
other protein delivery systems.

[0110]CTL activation increases observed for PPAA-PDSA-Ovalbumin conjugates
are summarized in Table 1. The fold increase in CTL activation is given
for the test sample, listed in the first column, when compared to the
control sample listed in the second column. Furthermore, p-values
resulting from ANOVA statistical analysis are provided in the final
column. p-values>0.05 are considered statistically insignificant. This
table clearly shows the advantages in CTL activation provided by
conjugation to PPAA.

[0111]Polymer Composition Cytotoxicity. The cytotoxicity of the PPAA-PDSA
and PMAA-PDSA polymers and conjugates was tested in both cell lines used
in this study: RAW macrophages and B3Z T-cells. Concentrations up to 300
μg/ml, twice the concentration used in the MHC-I presentation assay,
were tested using the LDH assay. This assay allows colorimetric
measurement of

[0112]LDH activity in the supernatant, which correlates to the proportion
of dead or damaged cells. Cell survival was calculated by comparing the
polymer-treated cells with untreated cells and with cells lysed with 1%
Triton X-100. It can be seen in FIG. 8 that none of the PPAA-PDSA or
PMAA-PDSA polymers or conjugates exhibit considerable toxicity in either
cell type at a concentration of 300 μg/ml.

[0113]A representative pH-sensitive, membrane-disruptive polymer,
poly(propylacrylic acid) (PPAA), was synthesized and used to enhance the
cytoplasmic delivery and MHC-I presentation of a model protein antigen,
ovalbumin. The protein was derivatized to have a thiol group, which was
reacted with the pendant PDSA disulfide bond on the polymer to yield a
polymer-protein conjugate linked by a disulfide bond. This
polymer-S--S-protein linkage was designed to allow release of the protein
by glutathione reduction in the cytoplasm. The polymer exhibited low
toxicity in vitro and retained its membrane-disruptive capabilities after
attachment to a hydrophilic protein, as indicated by a red blood cell
hemolysis assay. Conjugation to the PPAA polymer was shown to
significantly enhance the MHC-I presentation of ovalbumin and subsequent
CTL activation. This effect is a result of increased cytoplasmic delivery
of ovalbumin, which occurs due to destabilization of the endosomal
membrane by PPAA. These results suggest that the intracellular
pharmacokinetic step of vesicular release is a limiting barrier to
subsequent protein processing and MHC-1 presentation. This system thus
shows promise for protein vaccine strategies against cancer and viruses,
and is also applicable to any technique requiring improved delivery of a
protein cargo to the cytoplasm of a cell.

[0115]In order to further elucidate the enhanced CTL activation provided
by the PPAA carrier and investigate the carrier's cytosolic delivery
capabilities, the cellular uptake and exocytosis of radiolabeled
ovalbumin with and without the carrier was evaluated. Several methods
have been employed for conducting similar studies on intracellular
trafficking of molecules. For example, fluorescently labeled molecules
are followed using either confocal microscopy or flow cytometry, and
these studies often employ lysosomal, nuclear, or outer membrane
fluorescent markers to more specifically determine the molecule's
localization. Radiolabeled compounds have also been employed to study
cellular trafficking, sometimes in combination with subcellular
fractionation techniques to obtain detailed localization information.
14C radiolabels were chosen for this study due to their high
sensitivity and stability.

[0116]Cellular uptake and exocytosis of 14C-labeled ovalbumin was
quantified in a RAW macrophage cell line. Delivery of ovalbumin
conjugated to the endosome-disruptive PPAA carrier was compared to
delivery of free ovalbumin, a physical mixture of PPAA and ovalbumin, and
ovalbumin conjugated to non-active PMAA. The overall uptake of
14C-ovalbumin was measured after various incubation times by
removing un-internalized ovalbumin and lysing the cells to measure the
amount of intracellular ovalbumin. Enhanced intracellular accumulation of
the PPAA-ovalbumin conjugate was observed. This can likely be attributed
to continued escape of the PPAA-ovalbumin into the cytosol, whereas the
unconjugated or PMAA-conjugated ovalbumin remains in the endo/lysosomal
compartment, where it can be exocytosed from the cell.

[0117]To further investigate this hypothesis, the experiment was performed
with several adjustments to specifically address exocytosis of these
compounds, utilizing a method similar to those employed previously for
the study of exocytosis of radiolabeled sucrose in guinea pig alveolar
macrophages, hapten-protein conjugates in murine macrophages, and PLGA
nanoparticles in vascular smooth muscle cells. The exocytosis of
14C-ovalbumin was measured by quantifying the amount of
radioactivity re-appearing in the cell supernatant after removal of
un-internalized ovalbumin. The PPAA-ovalbumin conjugates showed decreased
exocytosis and increased intracellular accumulation compared to controls,
which correlates very well with the enhancement in MHC-1 presentation and
CTL activation observed in the previous study.

[0118]Radiolabeled conjugates were synthesized and were of similar sizes
to the non-radioactive counterparts described above. See Example 2. The
14C-labeled PPAA conjugates had Mw=140 kD, Mn=40 kD,
PDI=3.5 and the PMAA conjugate had Mw=120 kD, Mn=50 kD,
PDI=2.4. The PPAA and PMAA conjugates were found to be 34 and 40 wt. %
ovalbumin, or 1.9 μg polymer per μg protein and 1.5 μg polymer
per μg protein, respectively.

[0119]In order to further explore the intracellular release provided by
PPAA conjugation, the cellular uptake and exocytosis of
14C-ovalbumin was studied in RAW macrophages. The intracellular
accumulation of 14C-ovalbumin over increasing incubation times was
measured for PPAA-ovalbumin conjugates as well as negative controls: free
ova, PPAA+ova physical mixtures, and PMAA-ova conjugates. The enhanced
intracellular accumulation of the PPAA-ova conjugate is demonstrated in
FIG. 9. After a 15-min incubation, the amount of PPAA-conjugated
ovalbumin inside the cell was already twice that of the control groups.
Between 15 min and 2 hrs, the amount of intracellular PPAA-ovalbumin
increased from 0.75% of the total amount delivered to 4.8%, whereas the
control groups only increased from around 0.35% of the total amount
delivered to 0.55%. These differences in cellular accumulation are likely
due to continued escape of the PPAA-ovalbumin into the cytosol, whereas
unconjugated or PMAA-conjugated ovalbumin remains in the endo/lysosomal
compartment where it can be exocytosed.

[0120]To investigate this hypothesis, the experiment was repeated with
several adjustments to specifically address exocytosis of these
compounds. The samples were incubated with RAW macrophages for 1 min,
then any un-internalized sample was removed by washing the cells 2×
with media. Fresh media was applied at regular time intervals from 5 min
to 4 hrs, followed by lysis of the cells. The amount of radioactivity
which reappeared in the supernatant at each time interval, as well as the
radioactivity remaining in the cell lysate, was measured. This method is
similar methods previously employed for the study of exocytosis of
radiolabeled sucrose in guinea pig alveolar macrophages, hapten-protein
conjugates in murine macrophages, and PLGA nanoparticles in vascular
smooth muscle cells. Following the short 1-min incubation time, the
initial amount of 14C-ovalbumin taken up by the cells was
statistically similar for all the sample groups (ANOVA p-value=0.66), and
was approximately 0.8% of the total amount delivered (FIG. 10A).

[0121]However, less PPAA-conjugated ovalbumin was exocytosed compared to
the control groups, which is detailed in the exocytosis profiles shown in
FIG. 10B. This resulted in greater accumulation of the PPAA-ovalbumin
inside the cells, even after 4 hours of exocytosis (FIG. 10C). This
figure shows that 52% of the internalized PPAA-ovalbumin was still inside
the cells at the end of the experiment, whereas less than 10% of the
ovalbumin remained for all the control samples, including the non
membrane-active PMAA conjugate. This strongly supports the hypothesis
that PPAA-ovalbumin accumulates in the cell as a result of decreased
exocytosis. This effect is expected based on the ability of the PPAA
polymer to disrupt the endosomal membrane and deliver the ovalbumin to
the cytosol before exocytosis can occur.

[0122]These results further support the assertion that increased MHC-1
presentation and CTL activation is afforded by PPAA conjugation due to
enhanced cytosolic delivery of ovalbumin, and is not solely a result of
increased compound size. It can be noted that for all samples,
reappearance of the radiolabeled ovalbumin in the supernatant was
observed as early as 5 min after the cells were washed, and the majority
of exocytosis occurred in the first 30 min, a timescale which is in
accordance with previous exocytosis studies. Due to the negative charge
of both the PPAA and PMAA polymers at physiological pH, it is highly
unlikely that these results are affected by compounds sticking to and
subsequently releasing from the cell's negatively charged outer membrane.

[0123]In order to verify the trends recorded in this experiment, and to
reduce variability, the experiment was repeated using a longer 15-min
initial incubation time of the radiolabeled compounds with the cells. The
preferential intracellular accumulation of PPAA-conjugated ovalbumin can
already be seen in the initial cell uptake at this incubation time (14%
for PPAA-ovalbumin vs. 3% for control samples, FIG. 11A). However, after
normalizing to the amount of 14C-ovalbumin internalized by the cells
at the start of the exocytosis measurements, the rates of exocytosis
(FIG. 11B) and the amounts of ovalbumin remaining in the cells at the end
of the experiment (44% for PPAA-ovalbumin vs. 10% for control samples,
(FIG. 11c) are shown to enforce the trends observed for the 1 min uptake
time.

[0124]Cellular uptake and exocytosis of 14C-labeled ovalbumin was
quantified in a RAW macrophage cell line. Ovalbumin conjugated to PPAA
showed decreased exocytosis and increased intracellular accumulation
compared to controls, which correlates very well with the enhancement in
MHC-1 presentation and CTL activation observed. This can likely be
attributed to continued escape of the PPAA-ovalbumin into the cytosol,
whereas the unconjugated or PMAA-conjugated ovalbumin remains in the
endo/lysosomal compartment, where it can be exocytosed from the cell.

[0125]Formulation and In Vitro Evaluation of Poly(Propylacrylic
Acid)-Ovalbumin/Poly(dimethylaminoethylmethacrylate) Ionic Particles

[0126]Several studies have shown that particulate substances undergo
increased uptake and cross presentation by antigen presenting cells than
do soluble substances. In order to investigate whether a particulate
formulation of our PPAA carrier would result in greater delivery
efficiency, poly(dimethyl amino ethyl methacrylate) (pDMAEMA) was
employed as a cationic complexing agent, which resulted in the formation
of ionic particles 100-300 nm in size when combined with the anionic
PPAA-ovalbumin conjugate. Cellular uptake and exocytosis of these
particles to their soluble counterparts in a RAW macrophage cell line,
and it was found that the particulate formulation was indeed
advantageous. The ability of the carriers to enhance MHC1 presentation of
ovalbumin was then evaluated in another cell line, the mouse dendritic
cell line DC2.4. This cell line was employed as it was desired to test
the carrier in a more dendritic-like cell line. The DC2.4 line was
derived from murine bone marrow cells transfected with myc and raf
oncogenes as well as the granulocyte macrophage colony stimulating factor
(GM-CSF) gene. These cells possess the highly phagocytic nature of
immature DCs, as well as the MHC presentation and expression of other
surface markers characteristic of mature DCs. This line can be used in
coordination with the B3Z CTL hybridoma, which produces B-galactosidase
when activated, and has been used in previous studies for the evaluation
of PLGA-based vaccine delivery systems. It was found that the PPAA
particles resulted in substantially greater MHC1 presentation of
ovalbumin than did the soluble conjugates, which indicates that either
the uptake or cross-presentation, or both, are increased for particulate
carriers in these cells.

[0127]The PPAA-PDSA and PMAA-PDSA polymers described above were used to
form the ovalbumin conjugates. These conjugates were of a similar size to
those used described above: Mw=118 kD, Mn=44 kD, PDI=2.7 for
the PPAA-PDSA-ovalbumin conjugate and Mw=120 kD, Mn=55 kD,
PDI=2.3 for the PMAA-PDSA-ovalbumin conjugate. GPC analysis of pDMAEMA in
DMF using PMMA standards gave Mw=9.4 kD, Mn=7.8 kD, PDI=1.2.

[0128]The particle formulation process is described in Example 3 and shown
schematically in FIG. 12. The process proved to be very reproducible, and
representative particle sizes and zeta potentials are given in Table 2
for several -/+charge ratios. All particle sizes are reported as the
number average diameter. The charge ratios shown are theoretical,
calculated from the amounts and sizes of the negatively charged PPAA and
positively charged pDMAEMA and their expected degrees of ionization at pH
7.4. It can be seen that -/+charge ratios between 60:1 and 15:1 give
particles in the target range of 100-300 nm. Charge ratios near 1:1
result in the formation of large aggregates. The zeta potentials were
strongly negative for all the charge ratios. Since the zeta potential is
a measure of the charge on the particle surface, it is not necessarily
expected to increase with an increase in + charge in this range, as the
overall ratio is still largely negative. Positively charged particles
were not used, as the amount of pDMAEMA necessary to shift the net
particle charge to positive was very great and resulted in excessive
toxicity and adherence to cells in preliminary in vitro studies.

[0129]The toxicity of PPAA-Ova and PMAA-Ova conjugates and their
corresponding pDMAEMA particles was tested after a 24-hour incubation
with RAW macrophages, using the Alamar Blue assay. This assay utilizes a
fluorescent redox indicator to quantify cell growth and metabolism, and
can therefore be used to measure cytotoxicity. The particles were shown
to cause no appreciable toxicity at polymer concentrations up to 750
μg/ml, 5× that used in the cellular uptake and MHC-1
presentation assays (FIG. 13). The slight increase in cell viability
observed in the figure may be a result of the conjugates containing
ovalbumin, which the cells could use as a nutrient. This result is
expected based on previous results showing PPAA and PMAA polymers to be
nontoxic in an LDH cytoxicity assay (section 2.3.4). In this experiment,
the particles were established as nontoxic, which is important given
their larger size and different architecture compared to the soluble
polymers.

[0130]The uptake and exocytosis of the PPAA-Ova/pDMAEMA particles was
compared to the soluble conjugates in a RAW macrophage cell line as
described in Example 3. The amount of internalized 14C-Ova was
measured after incubation times of 15 min, 30 min, 1 hr, and 2 hr, and
the results are depicted in FIG. 14. It can be seen that the PPAA
particle does indeed result in increased intracellular accumulation
compared to the soluble PPAA conjugate. This effect becomes more
pronounced with increasing uptake time: uptake of the PPAA particle is
1.5× that of the PPAA conjugate after 15 min of uptake, and
increases to 3.6× after 2 hrs. It can also be seen that the
non-membrane active PMAA particle shows increased uptake over the soluble
PPAA conjugate, mainly at the longer uptake times. This suggests that
particulates accumulate more efficiently than soluble substances in this
cell type, and that this effect is not dependent on membrane-disruptive
activity of the polymer. This may be due to a more efficient uptake
mechanism for particulate substances through phagocytosis, which has been
report to be very highly efficient. Also, a different, lengthier
mechanism is implicated since the particles show preferential
accumulation primarily at the longer incubation times (>1 hr), but not
the shorter incubation times (<30 min). An intrinsic cytosolic
delivery pathway has been suggested for uptake by the processes of
phagocytosis, and it has been shown that latex beads taken up by
phagocytosis start to appear in the cytosol 1-2 hours after exposure to
macrophages.

[0131]If there are indeed different uptake efficiencies or uptake
mechanisms involved for the particulate vs. soluble conjugates, it may be
difficult to completely separate the particle-induced change in
uptake/processing from PPAA's endosomal release effects. It is important
to note that the intracellular accumulation of the PPAA particle is 45%
greater than that of the PMAA particle of the same size, even at the 2 hr
timepoint. The PPAA particles, therefore, may benefit from a combined
effect of increased uptake/cytosolic delivery due to their particulate
nature and enhanced cytosolic delivery due to endosomal escape provided
by the polymer. While soluble and particulate carriers are both
beneficial, depending on their application, a pH-sensitive particulate
carrier may prove to be the most optimized vehicle for immunotherapeutic
strategies.

[0132]To further explore the uptake and retention of these soluble vs.
particulate substances, the exocytosis of the substances was followed as
described in Example 3. The samples were incubated with RAW macrophages
for 15 min, then any un-internalized sample was removed by washing the
cells 2× with media. Fresh media was applied at regular time
intervals from 5 min to 4 hrs, followed by lysis of the cells. The amount
of radioactivity which reappeared in the supernatant at each time
interval, as well as the radioactivity remaining in the cell lysate, was
measured. Increased intracellular accumulation and decreased exocytosis
was observed for PPAA particles and soluble conjugates, and to a lesser
extent for PMAA particles.

[0133]The exocytosis profiles for all samples are shown in FIG. 15A, with
the amount of 14C-ovalbumin remaining in the cells at the conclusion
of the exocytosis study summarized in FIG. 15B. Most exocytosis occurs in
the first 5-10 minutes. It can be seen in FIG. 15B that both the PPAA
particle and conjugate show greater retention/reduced exocytosis than
does the PMAA particle (p<1×10-6). After 4 hours of
exocytosis, 55% of the internalized PPAA particle and 47% of the
internalized PPAA conjugate, respectively, remain inside the cell,
whereas only 33% of the PMAA particle remains inside the cell. The amount
is even lower for the free ovalbumin (14%) and soluble PMAA conjugate
(18%).

[0134]The amount of initially internalized ovalbumin at the beginning of
this exocytosis study is greater for the PPAA conjugate and particle than
the other samples (FIG. 16). This is in agreement with the studies
discussed in section 3.3.3 showing decreased exocytosis of PPAA-ova
starting at very early timepoints, which even by 15 min can result in a
noticeable excess of PPAA-conjugated ovalbumin in the cell.

[0135]When a very short 1 min uptake time was used, the initial amount of
14C-ovalbumin taken up by the cells was statistically similar for
all the sample groups (p=0.66). Furthermore, at the short 1 min uptake
time, the non membrane-active PMAA particle did not result in increased
intracellular retention (after 4 hrs exocytosis) compared to the free
ovalbumin and PMAA soluble conjugate controls (p>0.39), whereas the
PPAA was able to provide enhanced cellular retention of internalized
substances in both the soluble and particulate form (p<0.03) (data not
shown). The difference in retention observed with the PMAA particle at
the 1 min and 15 min uptake times could be due to a longer
uptake/processing time required for particulate matter.

[0136]The antigen presentation of ovalbumin delivered in PPAA ionic
particles was evaluated by the lacZ antigen presentation assay as
described in Example 3 using the dendritic cell line DC2.4 as the antigen
presenting cells. It was found that the particles did enhance MHC-1
presentation/CTL activation when compared to the free ovalbumin and to
the soluble PPAA conjugates, by about 15-fold (insert pvalue), as shown
in FIG. 17. Interestingly, the soluble conjugates did not result in
significantly increased presentation over delivery of free ovalbumin
(insert pvalue). This may be due to the nature of this cell line, which
has consistently shown increased phagocytosis and cross-presentation of
particulate substances compared to soluble substances for other materials
as well. The results of this study therefore indicate that the
PPAA-ovalbumin particles will be promising carriers for the delivery of
protein therapeutics in an in vivo vaccination model.

[0137]In this study, the PPAA conjugates were incorporated into a
particulate formulation by addition of an ionic complexing agent,
pDMAEMA. When the uptake and exocytosis of the particles was evaluated in
RAW macrophages, the intracellular accumulation of PPAA particles was
found to be greater than both the soluble PPAA conjugates and the PMAA
control particles. This suggests that they may benefit from a combined
effect of increased uptake/cytosolic delivery due to their particulate
nature and enhanced cytosolic delivery due to endosomal escape provided
by the polymer. The PPAA particles provided enhanced MHC-1 presentation
of ovalbumin in a dendritic cell line, compared to both free ovalbumin
and the soluble PPAA conjugate, and exhibited no toxicity in vitro. These
combined results indicate that the PPAA particles, in addition to the
soluble conjugates, show promise for in vivo vaccination applications.

[0139]Because PPAA is a promising carrier for protein vaccines in in vitro
studies, in both its soluble and particulate forms, its therapeutic
effect was evaluated in an in vivo model. A previously established murine
ovalbumin tumor model was employed. This model utilizes a tumor cell
line, E.G7-OVA, which is derived from the C57B1/6 mouse EL4 thymoma cell
line by electroporation with ovalbumin plasmid DNA. Since the tumor cells
are derived from the inbred C57B1/6 mouse strain, they can be injected
into fully immunocompetent C57B1/6 mice to form ovalbumin expressing
tumors. The tumor cells produce and process cytosolic ovalbumin,
presenting peptides on MHC1 molecules. Therefore, ovalbumin can be
delivered as a protein antigen in immunotherapeutic strategies. This
tumor model can provide valuable insight into carrier performance and has
been used by researchers to evaluate delivery systems utilizing
IL-2-containing liposomes, acid degradable particles, mannose-coated
liposomes, viral translocation motifs, cholera toxin, and various
adjuvants such as α-galactosylceramide, poly (I:C)/anti CD-40, and
imiquimod.

[0140]The therapeutic effect was evaluated in an in vivo model as
described in Example 4. Vaccines were injected 7 days prior to tumor cell
injection. Both soluble and particulate PPAA formulations were evaluated,
using PBS, free ovalbumin, and PMAA soluble and particulate carriers as
controls. In order to further explore the immune response, blood was
drawn from all mice and used to determine anti-ovalbumin IgG antibody
titer by ELISA. Finally, the number of ovalbumin reactive CD8+ T cells
present in the spleens of the mice was measured by flow cytometry using
MHC-1/SIINFEKL specific tetramers. These tetramers are fluorescently
labeled and bind to the T-Cell Receptor of CD8+ T-cells that are reactive
to ovalbumin. The results of the in vivo analysis showed that both the
soluble and particulate PPAA conjugates provided substantial tumor
protection compared to delivery of ovalbumin alone. Control mice which
received only PBS for the vaccine injection began to grow tumors 5 days
after tumor cell injection. Mice vaccinated with free ovalbumin began to
grow tumors 8 days after tumor cell injection and showed only slightly
slower growth compared to the PBS mice. All PBS and ovalbumin-injected
mice had to be euthanized by 19 days after tumor injection, as the tumors
exceeded a volume of 2 cm3. The mice receiving PPAA-ovalbumin
particulate formulations remained tumor free 4 weeks after tumor cell
injection, and only 1 mouse in the PPAA-ovalbumin soluble group had begun
to develop a tumor. These carriers were shown to result in increases in
both the antibody and CD8+ response, compared to controls. These
preliminary results suggest that PPAA is a promising carrier for in vivo
immunotherapy strategies.

[0141]The samples used in the in vivo studies are characterized in Table 3
below. The particle sizes reported are the number average. The PPAA-PDSA
and PMAA-PDSA polymers used to form the samples are described in the
examples.

[0142]The in vivo efficacy of PPAA as a vaccine carrier was evaluated
using a murine ovalbumin tumor model. This model utilizes the E.G7-OVA
tumor cell line, a derivative of the EL4 thymoma line which has been
transfected with the ovalbumin gene so that the tumors formed present
ovalbumin. These cells were derived from the same mouse strain used in
the study, C57B1/6, which ensures immunocompatibility of the tumor cells
themselves. This model has been previously employed for the
characterization of PLGA and acid-degradable particulate systems as well
as to evaluate various adjuvants and has proven a very informative
indicator of vaccine function.

[0143]The PPAA-ovalbumin and control vaccines were delivered
subcutaneously to the mouse's right flank 7 days prior to the injection
of the tumor cells subcutaneously to the left flank. This causes
formation of a tumor just beneath the skin which can be measured to
calculate the tumor volume. Tumor growth was monitored for 4 weeks. Both
the soluble and particulate PPAA carriers provided excellent tumor
protection, as evidenced by the tumor growth plots shown in FIG. 18.

[0144]Control mice which received only PBS for the vaccine injection began
to grow tumors 5 days after tumor cell injection. Mice vaccinated with
free ovalbumin began to grow tumors 8 days after tumor cell injection and
showed only slightly slower growth compared to the PBS mice. All PBS and
ovalbumin-injected mice had to be euthanized by 19 days after tumor
injection, as the tumors exceeded a volume of 2 cm3. Statistical
analysis performed on the tumor volumes at various timepoints show that
the tumor sizes were not significantly different for the PBS vs. free ova
mice (insert table of pvalues). After 4 weeks, all mice receiving the
PPAA-ovalbumin particle vaccine remained tumor free and only 1 mouse
which received the PPAA-ovalbumin soluble conjugate had begun to develop
a tumor. There was not an appreciable difference between the soluble and
particulate formulations.

[0145]The tumor protection provided by the PPAA carrier is very strong
when placed in the context of other vaccine systems utilizing this model.
Ovalbumin co-delivered with the adjuvant IL-12p40 provided protection for
9 days, ovalbumin delivered with IL-2 exosomes provided protection for 22
days, and ovalbumin delivered via IL-2-containing liposomes provided
protection for 45 days. The results indicate that the PPAA carrier can
stimulate an immune response capable of providing tumor protection.

[0146]In order to determine the role of the humoral immune response in the
tumor protection observed for PPAA-ova vaccinated mice, blood was drawn
from all mice 20 days after vaccination and the anti-ova IgG titer was
measured by ELISA. FIG. 19 shows the average antibody concentration per
group, as determined by an anti-ova IgG standard curve. No antibody
production was observed for PBS or free ova-vaccinated mice, whereas the
presence of anti-ova antibodies was detected for PPAA-ova vaccinated
mice. This indicates that an antibody-mediated response is implicated in
the tumor protection observed for these mice. Some degree of antibody
response is expected, and has been observed in additional studies which
also report enhanced CTL activation. The increased antibody response
observed for the PPAA-ovalbumin compared to delivery of ovalbumin alone
is likely a product of the enhanced stability and lengthened circulation
time provided by the polymer. It may also be indicative of an enhanced
CD4+ T-helper cell response, which occurs through the MHC2 antigen
presentation pathway, since one of the primary functions of T-helper
cells is to activate and support antibody production by B-cells.

[0147]In order to more closely investigate the role of enhanced CTL
activation in the observed tumor protective immune response,
MHC1/SIINFEKL tetramers were used to measure the number of ova-reactive
CD8+T cells in the spleen by flow cytometry. See Example 4. MHC-1
tetramers are composed of 4 MHC-1 molecules bound to the class I peptide
epitope, in this case the SIINFEKL peptide derived from ovalbumin, and
conjugated to a fluorescent molecule. These tetramers therefore bind to
the T-cell receptor of CD8+ T-cells that are reactive to ovalbumin. MHC-1
tetramers have been employed to evaluate CD8+ responses in vaccine
strategies using ovalbumin, as well as other antigens.

[0148]Mice (4 per group) were immunized with soluble and particulate
PPAA-ovalbumin conjugates 7 days prior to sacrifice/splenocyte harvest.
Mice immunized with either PBS, free ovalbumin, or PMAA soluble or
particulate carriers were used as controls, and the experiment was
performed 2 times independently. Delivery of free ovalbumin did not
result in statistically significant CTL activation compared to PBS
(p=0.31). Vaccination with soluble and particulate PPAA-ovalbumin
conjugates resulted in enhanced CTL activation compared to PBS
(p<0.0001), free ovalbumin (p<0.0002), and soluble PMAA-ovalbumin
(p<0.0001), as evidenced by the increased number of CD8+/tetramer+
T-cells depicted in FIG. 20. Particulate PMAA-ovalbumin provided
intermediate CTL activation. The CTL activation provided by the PMAA
particle is likely due to the increased uptake and cross-presentation
previously observed for particulate substances taken up by phagocytosis.
CTL activation was slightly increased for particulate PPAA compared to
soluble PPAA (p=0.29). An increase is expected based on the potential
advantage of the particulate formulation to benefit from both increased
uptake/presentation and cytosolic escape.

[0149]The ability of the PPAA protein antigen carrier to provide in vivo
tumor protection was evaluated. It was concluded that both the soluble
and particulate forms of the carrier provide strong protection against
ovalbumin-expressing tumors. The anti-tumor immune response was found to
consist of an increase in both anti-ova antibody production as well as
anti-ova CTL activation. The PPAA-ova vaccines were able to prevent tumor
growth for the duration of the 4-week study, with the exception of 1
mouse which had begun to develop at tumor on day 26, whereas control mice
injected with PBS and free ovalbumin all developed tumors and had to be
removed from the study by day 19.

[0150]Delivery of NY-ESO-1 Tumor Antigen to Primary Dendritic Cells Using
Poly(Propylacrylic Acid) and Evaluation of Class I Presentation

[0151]In addition to the delivery of whole protein antigens in
immunotherapeutic strategies, the specific MHC1 peptide epitope sequences
can be delivered. This approach is advantageous in that the peptides are
smaller in size and can be chemically synthesized, making them
potentially easier to deliver in higher concentrations, as well as
cheaper and more readily obtained than the full protein for most
clinically relevant antigens. PPAA was used to deliver a peptide antigen
to primary human dendritic cells (DCs). The peptide antigen to be
delivered is the Class I HLA-A2 epitope (residues 157-165) of the
NY-ESO-1 human tumor antigen, which is expressed by melanoma, ovarian,
breast, and other cancers. The peptide was conjugated to the PPAA-PDSA
polymer and delivered to primary human dendritic cells as described in
Example 5. The resulting Class I antigen presentation was evaluated using
an NY-ESO-1 (NY157-165)-specific CD8+ T cell clone. The PPAA carrier was
able to induce Class I presentation of a peptide antigen in a dose
dependent manner.

[0152]Polymer-peptide conjugation resulted in 80% of peptides reacting,
according to the pyridine 2-thione measure. This corresponds to a
conjugate composition of 14 wt % peptide and 86 wt % polymer. The
evaluation shown in FIG. 21 compared PPAA-conjugated SLLMCWITQC (SEQ ID
NO: 1) peptide with the free peptide, at increasing peptide
concentrations. Conjugates or peptides were incubated with immature DCs,
followed by DC maturation and incubation with HLA/peptide-specific CD8+
cells. The IFNγ produced by activated CD8+ cells was then measured
by ELISA.

[0153]It can be seen that the conjugate does result in CTL activation in a
dose-dependent manner, but not to the extent as that induced by the free
peptide alone. However, since the free peptide can load externally into
HLA molecules displayed on the DC surface, but the conjugate
theoretically cannot due to steric hindrance, it is difficult to directly
compare these two results. Another concern is that any unreacted peptide
that was not purified away from the conjugate could bind the HLA
externally and interfere with the results.

[0154]In order to address these issues, a second peptide sequence was
employed: CAARSLLMWITQV (SEQ ID NO: 2). The natural peptide sequence,
SLLMWITQC, was modified to give the alternate anchor residue valine. The
sequence has been further modified to contain a cysteine residue, used
for conjugation to the PDSA moiety of the PPAA polymer, attached to the
peptide by a short linker sequence on the amine terminus end. The linker
sequence culminates with an arginine residue, chosen based on studies
reporting preferential cleaving after R residues by aminopeptidases in
the ER and cytosol. This sequence therefore requires intracellular
processing before it is able to bind the HLA molecule and induce CTL
activation. It is also advantageous in that the polymer does not have to
be conjugated to a residue that is part of the antigenic epitope. The
results obtained with this conjugate are shown in FIG. 22, using the
SLLMWITQV sequence as a positive control.

[0155]Some CTL activation is again seen, in a dose-dependent manner. The
nature of the peptide sequence used indicates that the CTL activation
seen is due to intracellular processing.

[0156]In a further attempt to distinguish between distinguish between
activation due to antigens that have been internalized and processed vs.
external peptide loading, the ER transport and vesicle transport
inhibitor BFA was utilized. The results of this experiment are shown in
FIG. 23. Some decrease in CTL activation was seen when the samples were
treated with BFA, but a considerable degree of activation still occurred.
Furthermore, reduction was observed to the same extent for the free
peptide as for the conjugate, giving inconclusive results that suggest
the method did not perform as expected. Also, the unconjugated
CAARSLLMWITQV peptide gave activation levels higher than those of the
conjugate.

[0157]The PPAA carrier was able to induce Class I presentation of a
peptide antigen in a dose dependent manner. While external pulsing of DCs
with free peptides is efficient for ex vivo strategies, in vivo delivery
of injected peptide antigens will benefit from the stabilizing and
concentrating effects provided by the carrier. The hydrophilic PPAA-PDSA
polymer also confers solubility to the hydrophobic peptides, which
normally are soluble only in organic solvents.

[0158]Protein immunotherapy is an important emerging strategy in the
treatment of cancers. It offers significantly reduced toxicity compared
to radiation and chemotherapy, is more tunable to specific
cancers/patients, and provides a new means to combat cancers which show
resistance to conventional treatments. Ongoing investigations in this
area have revealed several opportunities for improving the efficacy of
protein immunotherapies.

[0159]The present invention addresses the challenge of increasing protein
antigen delivery to the cytosol of antigen presenting cells, where
antigens are able to more efficiently induce a potent anti-tumor CTL
response. Carriers based on the pH-sensitive, endosomally-active polymer
poly(propylacrylic acid) were formulated to deliver the model protein
antigen ovalbumin. The protein was conjugated through a disulfide linkage
which is reversible by the cytosolic gluthathione reduction system,
freeing the protein for the necessary processing. Both soluble and
particulate architectures of the carrier are provided. An increase in MHC
Class I presentation and subsequent CTL activation was demonstrated when
both a macrophage cell line and a dendritic cell line was used as the
antigen presenting cell. These increases in CTL activation were shown to
correspond with the ability of the PPAA polymer to provide reduced
exocytosis and thus increased intracellular accumulation over time.

[0160]These carriers also proved to provide excellent tumor protection in
vivo, enhancing both the antibody and CTL-based immune responses. The in
vivo advantages of the PPAA carrier are likely multi-faceted, as they not
only cytosolic escape, but are also likely to provide serum stability and
increased circulation time due to their hydrophilic and slightly
negatively-charged state at physiological pH. They also promote increased
uptake by APCs due to their larger size compared to delivery of free
antigen. No carrier toxicity was observed either in vitro or in vivo,
even at high concentrations, which is a key advantageous attribute. The
carrier is also applicable to the delivery of peptide vaccines.

[0161]The present invention demonstrates the efficacy of PPAA-based
carriers in the delivery of protein antigens for immunotherapeutic
strategies. The carriers are highly suited to in vivo vaccine
applications. Since the carrier has proven highly advantageous in the
delivery of the model antigen ovalbumin, it can be expanded to the
delivery of relevant human cancer antigens in mouse xenograft models.
Furthermore, the success of the carriers in these evaluations indicates
their applicability to a wide range of drug delivery processes which
necessitate the delivery of protein or peptide therapeutics to targets in
the cytosol of cells.

[0162]The following examples are provided for the purpose of illustrating,
not limiting, the invention.

EXAMPLES

General Methods and Materials

[0163]All chemicals and reagents were ACS grade purchased from
Sigma-Aldrich, St. Louis, Mo., and used without further purification
unless otherwise noted.

[0164]Polymer compositions were determined by H1--NMR using a Bruker
AVance 300 MHz instrument and deuterated dimethyl sulfoxide (DMSO-d6,
Fisher Chemical, Pittsburgh, Pa.). PDSA content was determined both by
NMR and by the absorbance at 343 nm of pyridine-2-thione released from
the polymer following reduction with excess of dithiothreitol (DTT). The
molecular weight distribution was determined by GPC (Viscotek VE2001
sample module, VE3580 RI Detector, Waters Corp. ultrahydrogel columns) in
0.1 M sodium phosphate buffer, pH 8 using poly(ethylene oxide) (PEO)
standards (Polysciences, Inc., Warrington, Pa.). The pKa of the PPAA-PDSA
was determined by acid/base titration of 50 mg polymer in distilled,
deionized water, starting at pH 12.5 and titrating with 0.1 N HCl.

[0165]Conjugates were characterized by GPC in 0.1M sodium phosphate buffer
pH 8 using PEG standards. The final weight percent of ovalbumin in the
conjugates was determined by the BCA protein assay (Pierce Biotechnology,
Rockford, Ill.), using an ovalbumin standard curve. SDS-PAGE
(polyacrylamide gel electrophoresis) was performed to detect any
unreacted ovalbumin in the conjugate solution. Precast Tris-HCl
polyacrylamide gels, 4-20% gradient, sample and running buffer, and
protein standards were purchased from Bio-Rad, Hercules, Calif. Based on
the results of the BCA assay, conjugates were loaded onto the gels in the
appropriate amounts to give 10 ug of ovalbumin per lane. Gels were run
for 45 min at 100 mA, then visualized using Coomassie staining (Bio-Rad,
Hercules, Calif.).

Example 1

Preparation and Characterization of a Representative pH-Responsive Polymer
Composition

PPAA-Ovalbumin

[0166]PPAA-PDSA. For the synthesis of the PPAA-PDSA polymer, 0.007 mol
propylacrylic acid (PAA) (Gateway Chemical Technology, St. Louis, Mo.),
0.00011 mol PDSA (synthesized according to previous protocols), and
0.000056 mol free-radical initiator azobisisobutyronitrile (AIBN,
purified by recrystallization from methanol) were combined in a 5 ml
flask and degassed by 4 rounds of freeze-vacuum-thaw then reacted at
60° C. for 24 hours. The polymer was dissolved in 3 ml dimethyl
formamide (DMF) and purified by 3 rounds of precipitation in 500 ml
diethyl ether.

[0168]Ovalbumin Conjugation. Conjugation was performed via disulfide
exchange between the PDSA component of the polymer and free thiols
introduced onto ovalbumin by reaction with Traut's reagent
(2-iminothiolane, Pierce Biotechnology, Rockford, Ill.). 10 mg ovalbumin
was mixed with a 10× molar excess of Traut's reagent in conjugation
buffer (0.1M phosphate buffer, pH 7.8, 0.15M NaCl, 5 mM EDTA) for 1 hour
at room temperature. The reaction mixture was purified using a PD-10
desalting column containing Sephadex G-25 (MWCO SkD, GE Healthcare,
Piscataway, N.J.) and the degree of modification was estimated by
Ellman's assay (Pierce Biotechnology, Rockford, Ill.). A 2.5× molar
excess of polymer, either PPAA-PDSA or PMAA-PDSA, was immediately added
to the modified protein and allowed to react 2 hours at room temperature
in conjugation buffer. The degree of conjugation was estimated by
measuring the absorbance at 343 nm (A343) of the pyridine-2-thione
group released from PDSA upon disulfide exchange, and the conjugate was
purified on a PD-10 column and lyophilized for storage.

[0169]The membrane-disruptive PPAA-PDSA polymer was synthesized by free
radical polymerization and resulted in a polymer with 3 mol % PDSA and
Mw=26 kD, Mn=10 kD, and PDI=2.6, based on GPC analysis using
PMMA standards. The pKa of this polymer was determined by acid/base
titration to be 6.8, which is in the range of the transition between
physiological and endosomal pH. A non membrane-disruptive polymer,
PMAA-PDSA, of similar size (Mw34 kD, Mn=12 kD, PDI=2.8)
containing 2 mol % PDSA was synthesized as a control. Methacrylic acid is
less hydrophobic than propylacrylic acid in the protonated form and does
not cause any membrane disruption in the RBC hemolysis assay.

[0170]The conjugation reaction can be controlled by adjusting the degree
of protein thiolation and the duration of the conjugation reaction. A
greater number of thiols on the protein, such as 5 or more, results in
crosslinking and larger conjugates with complex structures. However, when
the protein is modified to give an average of only 1-3 thiols per
protein, conjugation does not occur as extensively. The degree of
thiolation of the ovalbumin used to form the polymer-ovalbumin conjugates
was determined by Ellman's assay to be an average of 3 per protein, or
15% of the total available lysines.

[0171]A BCA assay was performed to determine the final weight % of
ovalbumin in each conjugate, using an ovalbumin standard curve, so that
the samples could be normalized to contain the same amount of ovalbumin
in the MHC-1 presentation assay. Both the PPAA-PDSA and PMAA-PDSA were
found to contain 37 weight % ovalbumin and 63 weight % polymer, or 1.7
μg polymer per μg protein. GPC analysis gave Mw=200 kD,
Mn=45 kD, PDI=4.4 for the PPAA-PDSA-ovalbumin conjugate and
Mw=130 kD, Mn=57 kD, PDI=2.3 for the PMAA-PDSA-ovalbumin
conjugate. SDS-PAGE was performed to ascertain that no free protein
remained in the conjugate mixtures, as it could interfere with
interpretation of the MHC-1 presentation assay results.

[0172]pH-Dependent Membrane-Disruptive Ability. The pH-dependent
membrane-disruptive ability of the polymers and polymer-protein
conjugates was estimated using a red blood cell hemolysis assay. Briefly,
red blood cells were isolated and added to polymer and conjugate
solutions (normalized to equivalent polymer amounts) of varying
concentrations in 0.1M phosphate buffer at pH values of 5.8, 6.6, and
7.4. The degree of RBC membrane disruption (% hemolysis) was quantified
by measuring the absorbance at 541 nm of the hemoglobin released into the
solution by lysed cells, in comparison with complete lysis by Triton
X-100 detergent. Sample concentrations were all normalized to contain 5
μg/ml of polymer, and samples were performed in triplicate with error
reported as +/-one standard deviation.

[0173]MHC Class I Antigen Presentation Assay. The ability of the polymer
to increase cytoplasmic delivery and subsequent MHC class I antigen
presentation was evaluated using the lacZ antigen presentation assay.
This assay utilizes a specialized LacZ B3Z CTL hybridoma. These CTLs
produce β-galactosidase upon recognition of the ovalbumin class I
antigenic epitope SIINFEKL complexed with the MHC class I molecule
H-2Kb, present on RAW 309.1 CR macrophages. Therefore, a measure of
β-galactosidase activity can be used to determine the degree to
which delivered ovalbumin is presented as a class I antigen. All tissue
culture reagents were purchased from Invitrogen Corp, Carlsbad, Calif.,
unless otherwise noted. RAW 309.1 CR macrophages (ATCC, Manassas, Va.)
were cultured in 90% DMEM with D-Glucose and L-glutamine, 10% fetal
bovine serum (FBS), supplemented with 100 U/ml penicillin/100 μg/ml
streptomycin. B3Z CTLs were a gift from Dr. Nilabh Shastri, UC Berkeley.
They were cultured in 90% RPMI medium with D-glucose and L-glutamine, 10%
FBS, supplemented with 100 U/ml penicillin/100 μg/ml streptomycin, 50
μM 2-mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.), and 1 mM sodium
pyruvate. For the assay, RAW cells were plated at 5×104 cells
per well in a 96-well plate and grown overnight. PPAA-ovalbumin
conjugates and control samples were added to the cells and incubated 6
hrs in DMEM with 5% FBS. Three sample concentrations were tested: 50
μg/ml, 100 μg/ml, and 150 μg/ml (samples were normalized to
represent equivalent ovalbumin concentrations). RAW cells were rinsed
with DMEM, then 1×105 B3Z cells per well were added and
incubated 16 hrs. Cells were rinsed with phosphate buffered saline then
100 μl lysis buffer [100 μM mercaptoethanol (Sigma-Aldrich, St.
Louis, Mo.), 9 mM MgCl2 (Sigma-Aldrich, St. Louis, Mo.), and 0.15 mM
chlorophenol red β-D-galactoside (EMD Biosciences, San Diego,
Calif.) in PBS] was added. After 4 hrs the absorbance of released
chlorophenol red was measured at 595 nm. All samples were evaluated in
triplicate and errors are reported as +/-one standard deviation.
Statistical results (Student's t-test) are reported for the 100 μg/ml
concentrations. The maximum possible β-galactosidase production was
determined by chemically stimulating the B3Z cells in media containing
3.15 μM ionomycin (Sigma-Aldrich, St. Louis, Mo.) and 10 ng/ml phorbol
12-myristate 13-acetate (Sigma-Aldrich, St. Louis, Mo.) for 4 hours
before rinsing and adding the lysis buffer.

[0174]Cytotoxicity Assay. The cytotoxicity of the PPAA and PMAA polymers
and conjugates was determined for both the RAW and B3Z cell lines used in
the MHC-1 presentation assay. Cytotoxicity was evaluated using the LDH
(lactate dehydrogenase) assay (Roche Applied Sciences, Indianapolis,
Ind.). This assay allows colorimetric measurement of LDH activity in the
supernatant, which correlates to the proportion of dead or damaged cells.
Cells were plated at 5×104 cells/well in their normal culture
media and polymer and conjugate samples were added to concentrations up
to 300 μg/ml then incubated for 24 hrs. Cells were centrifuged at 250
g for 10 min, then 100 μl supernatant was removed and combined with
100 μl LDH reagent. The absorbance at 490 nm (reference 650 nm) was
recorded every 5 min for 30 min and cell survival was determined by
comparing to untreated cells and cells lysed with 1% Triton X-100 in
water. Each sample was evaluated in triplicate and errors are reported as
the standard error of the mean (SEM).

Example 2

Preparation and Characterization of 14C-Ovalbumin Conjugates

[0175]Preparation of PPAA/PMAA-14C-Ovalbumin Conjugates.
Radioactively labeled ovalbumin-PPAA-PDSA and PMAA-PDSA conjugates were
formed for tracking of the ovalbumin in cellular uptake and exocytosis
experiments. The procedure used was similar to that described above for
conjugate synthesis, except that a 14C label was added to ovalbumin.
Ovalbumin was mixed with a 20× molar excess of Traut's reagent,
followed by a 3× molar excess of 14C-iodoacetamide (MP
Biomedical, Solon, Ohio). After reacting for 1 hour, a 2.5× molar
excess of polymer, either PPAA or the negative control polymer PMAA, was
added. Conjugates were again characterized by GPC in 0.1M sodium
phosphate buffer pH 8 using PEG standards, and the final weight percent
of ovalbumin was determined by a BCA assay. The amount of 14C per
conjugate was determined by liquid scintillation counting, using a
Beckman-Coulter LS600 liquid scintillation counter and EcoScint
scintillation fluid (National Diagnostics, Atlanta, Ga.).

[0176]Cellular Uptake of PPAA-14C-Ovalbumin Conjugates. The cellular
uptake and accumulation of 14C-labeled ovalbumin and the PPAA and
PMAA-ovalbumin conjugates was studied in RAW 309.1 CR macrophages. Cells
were plated in a 48-well plate at 75,000 cells/well and allowed to grow
overnight. Either ovalbumin, PPAA-ovalbumin conjugate, PMAA-ovalbumin
conjugate, or a PPAA and ovalbumin physical mixture was added to the
cells at a concentration of 50 μg/ml of ovalbumin. Samples were
incubated for either 15 min, 30 min, 1 hr, or 2 hrs. The cells were then
washed 2× with PBS and lysed using 1% Triton X-100 in water.
Radioactivity in the cell media, PBS wash, and cell lysate was measured
using a Beckman-Coulter LS 6500 liquid scintillation counter. EcoScint
scintillation fluid was obtained from National Diagnostics, Atlanta, Ga.
Uptake of 14C-ovalbumin is presented as the % radioactivity present
in the cell lysate compared to the total radioactivity delivered. The
experiment was performed with a minimum of n=3.

[0177]Exocytosis Profiles of PPAA-14C-Ovalbumin Conjugates. The
exocytosis of 14C-ovalbumin was measured using a procedure similar
to that described by Besterman et al. RAW macrophages were plated at
75,000 cells/well in a 48-well plate and allowed to grow overnight.
Either ovalbumin, PPAA-ovalbumin conjugate, PMAA-ovalbumin conjugate, or
a PPAA and ovalbumin physical mixture was added to the cells at a
concentration of 50 μg/ml of ovalbumin. Samples were incubated for
either 1 min or 15 min, then uninternalized conjugate was removed and
cells were washed 2× with media. Fresh media was added to the cells
at the following timepoints: 5 min, 10 min, 20 min, 30 min, 1 hr, 2 hr,
and 4 hr. The reappearance of 14C-ovalbumin into the supernatant was
measured at each timepoint. After 4 hrs, cells were lysed with 1% Triton
X-100 and the radioactivity in the lysate was measured. The amount of
ovalbumin internalized after the 1 min or 15 min incubation time was
determined as a percentage of the total delivered. The amount of
ovalbumin exocytosed at each timepoint, as well as the amount remaining
in the cells after 4 hrs, was then determined as a percentage of the
total internalized. The experiments were performed with a minimum of n=3.

Example 3

Formulation and Characterization of Representative Ionic Particles

PPAA-Ovalbumin/pDMAEMA

[0178]Ionic Particle Formation. Particles were formed by ionic
complexation of the cationic pDMAEMA with the anionic PPAA-Ova conjugate.
Conjugates were formed according to the procedures outlined in Examples 1
and 2. Briefly, ovalbumin was mixed with Traut's reagent, which reacts
with lysine amines to give reactive SH groups. 10-15% of the 20 available
lysines were modified. In the case of the radiolabeled conjugates used
for the uptake/exocytosis studies, 30% of the amines were modified, to
provide additional sites for attachment of the 14C label. The
ovalbumin-SH was purified on a PD-10 desalting column, then reacted with
the PDSA moiety on the PPAA or PMAA polymer. The conjugate was purified
and exchanged to PBS using a Zeba desalting spin column, then stored at
4° C., omitting the lyophilization and freezing processes
previously used. pDMAEMA was synthesized by RAFT (reversible addition
fragmentation chain transfer) radical polymerization. DMAEMA monomer was
dissolved in DMF at 33 wt. %. The chain transfer agent (CTA)
4-cyano-4-(ethylsulfanylthiocarbonyl)sulfanyl pentanoic acid (ECT) and
initiator 2,2'-azobis(4-methoxy-2,4-dimethylvaleronitrile) (V-70) were
added at a CTA:initiator ratio of 10:1. The solution was degassed with
N2 for 30 min, then reacted at 30° C. for 16 hrs and
precipitated 3× in diethyl ether.

[0179]To form the particles, Ova-PPAA or PMAA conjugates were dissolved in
PBS, pH 7.4. pDMAEMA was added at varying charge ratios calculated based
on the % ionization and molar quantity of the pDMAEMA and PPAA or PMAA.
Charge ratios ranging from neg:pos 40:1 to 1:3 were tested. The solution
was vortexed then allowed to sit for 1 hour at room temperature for
particle formation to occur. The particle size and zeta potential were
determined in PBS using a Brookhaven (Holtsville, N.Y.) BI90Plus
instrument equipped with a 535 channel correlator. Measurements were
performed at a 90° angle with a 656 nm laser as the incident beam,
and particle sizes are reported as the number average. Particle size
standard deviations were calculated from the reported polydispersity
according to the formula:

fwhm=Dh μ2

[0180]where Dh and μ2 are the hydrodynamic diameter and
polydispersity, respectively, reported by the particle sizer, and the
fwhm (full-width at half maximum) is related to the standard deviation
by: stdev=fwhm/[2 (21n2)].

[0181]In vitro Cytotoxicity of Ionic Particles. The cytotoxicity of
PPAA-Ova and PMAA-Ova/pDMAEMA particles was tested in RAW macrophages
using the Alamar Blue Cytoxocity Assay (Invitrogen, Carlsbad, Calif.).
Alamar Blue allows quantification of cell viability by utilizing an
oxidation-reduction indicator that fluoresces as growth medium undergoes
chemical reduction resulting from cellular growth and metabolism.
Particles were formed and characterized according to the procedure
described in section 3.2.3. RAW macrophages were plated at 25,000 cells
per well in 150 μl media (DMEM supplemented with 10% FBS) in a 96-well
plate and incubated for 30 min at 37° C. 200 Alamar Blue reagent
was added to each well. Then, Ova-PPAA and Ova-PMAA conjugates and
particles were added to the cells at polymer concentrations of 100
μg/ml, 200 μg/ml, 500 μg/ml, and 750 μg/ml. Triton X-100 was
used as a positive control for toxicity. Samples were incubated for 24
hrs, then fluorescence was measured using a Tecan Safire II plate reader
using the following settings: Excitation: 545 nm w/bandwidth 6 nm,
Emission: 590 nm w/bandwidth 20 nm, Gain: 68. % Survival compared to
untreated cells was calculated for each sample. Samples were evaluated in
triplicate and ANOVA statistical analysis was performed.

[0182]Cellular Uptake and Exocytosis of Ionic Particles. The uptake and
exocytosis of particulate Ova-PPAA and PMAA conjugates into RAW
macrophages was compared to their soluble counterparts according to the
procedure described in sections 3.2.2 and 3.2.3. For uptake studies,
cells were plated and grown overnight. The samples (ovalbumin,
PPAA-ovalbumin conjugate, PMAA-ovalbumin conjugate, and PPAA-ovalbumin
and PMAA-ovalbumin particles) were added to the cells at a concentration
of 50 μg/ml of ovalbumin. Samples were incubated for 15 min, 30 min, 1
hr, or 2 hrs. The cells were then washed 2× with PBS and lysed,
then radioactivity in the cell media, PBS wash, and cell lysate was
measured. Uptake of 14C-ovalbumin is presented as the %
radioactivity present in the cell lysate compared to the total
radioactivity delivered. The experiment was performed with a minimum of
n=3.

[0183]For exocytosis studies, cells were again plated and allowed to grow
overnight. The samples (ovalbumin, PPAA-ovalbumin conjugate,
PMAA-ovalbumin conjugate, and PPAA-ovalbumin and PMAA-ovalbumin
particles) were added to the cells at a concentration of 50 μg/ml of
ovalbumin. Samples were incubated for either 1 min or 15 min, then
uninternalized conjugate was removed and cells were washed 2× with
media. Fresh media was added to the cells at 5 min, 10 min, 20 min, 30
min, 1 hr, 2 hr, and 4 hr, and the reappearance of 14C-ovalbumin
into the supernatant was measured. After 4 hrs, the cells were lysed and
the radioactivity in the lysate was measured. The amount of ovalbumin
internalized after the 1 min or 15 min incubation time was determined as
a percentage of the total delivered. The amount of ovalbumin exocytosed
at each timepoint, as well as the amount remaining in the cells after 4
hrs, was then determined as a percentage of the total internalized. The
experiments were performed with a minimum of n=3.

[0184]MHC-1 Antigen Presentation and CTL Activation Assay in a Dendritic
Cell Line. The ability of the particles to increase MHC class I antigen
presentation in dendritic cells was evaluated using the lacZ CTL
activation assay. The B3Z CTLs produce β-galactosidase upon
recognition of the ovalbumin class I antigenic epitope SIINFEKL complexed
with the MHC class 1 molecule H-2Kb, which is present on the DC2.4
cells. DC2.4 cells were cultured in 90% DMEM with D-Glucose and
L-glutamine, 10% fetal bovine serum (FBS), supplemented with 100 μg/ml
penicillin/100 μg/ml streptomycin. B3Z CTLs were a gift from Dr.
Nilabh Shastri, UC Berkeley. They were cultured in 90% RPMI medium with
D-Glucose and L-glutamine, 10% FBS, supplemented with 100 U/ml
penicillin/100 μg/ml streptomycin, 50 μM 2-mercaptoethanol
(Sigma-Aldrich, St. Louis, Mo.), and 1 mM sodium pyruvate. For the assay,
DC2.4 cells were plated at 5×104 cells per well in a 96-well
plate and grown overnight. PPAA-ovalbumin conjugates and particles were
added to the cells at an ovalbumin concentration of 100 μg/ml and
incubated 6 hrs. Free ovalbumin was also tested at 100 μg/ml as well
as a high concentration of 5 mg/ml. The cells were rinsed with DMEM, then
1×105 B3Z cells per well were added and incubated 16 hrs.
Cells were rinsed with PBS then 100 μl lysis buffer [100 μM
mercaptoethanol (Sigma-Aldrich, St. Louis, Mo.), 9 mM MgCl2
(Sigma-Aldrich, St. Louis, Mo.), and 0.15 mM chlorophenol red
β-D-galactoside (EMD Biosciences, San Diego, Calif.) in PBS] was
added to each well. After 2 hrs the absorbance of released chlorophenol
red was measured at 595 nm. All samples were evaluated in triplicate and
errors are reported as +/-one standard deviation.

[0185]Sample Formulation. Soluble and particulate PPAA and PMAA ovalbumin
conjugates were formulated to give a final concentration of 100 μg
ovalbumin in a 150 μl injection volume. PPAA and PMAA conjugates and
particles were freshly prepared according to the procedure described in
Examples 2 and 3. A calculated -/+ charge ratio of 20:1 was used for
particle formation. The size of the conjugates was determined by GPC and
the size of the resulting pDMAEMA ionic particles was measured by DLS.

[0186]Tumor Protection Against Ovalbumin Expressing Tumors. The ability of
PPAA-ovalbumin vaccines to prevent growth of ovalbumin expressing tumors
was tested in mice. All studies were performed in compliance with the
University of Washington Animal Care and Use Committee. Female C57B1/6
mice 7-8 weeks old were purchased from Jackson Labs (Bar Harbor, Me.).
E.G7-OVA cells, which are derived from the same C57B1/6 inbred mouse
strain, were obtained from ATCC (Manassas, Va.) and cultured in RPMI 1640
containing 10 mM Hepes, 1 mM sodium pyruvate, 4.5 g/L glucose, and 1.5
g/L sodium bicarbonate (ATCC, Manassas, Va.) supplemented with 0.05 mM
2-mercaptoethanol (Sigma), 0.4 mg/ml G418 (Invitrogen), and 10% FBS
(Invitrogen). Mice were anesthetized with isofluorane and injected
subcutaneously on the right flank using a 27 gauge needle. Mice (a
minimum of 4 per group) were injected with 150 μl of PBS, ova, soluble
PPAA-ova, particulate PPAA-ova, soluble PMAA-ova, or particulate
PMAA-ova, with an equivalent of 100 μg ova delivered to each mouse.
Seven days after vaccine administration, mice were again anesthetized and
the hair on the left flank was removed using Nair. The E.G7-OVA tumor
cells (1×106 cells per mouse) were then injected in a volume
of 1000 PBS subcutaneously into the left flank. Mice were monitored every
2-3 days and tumor length and width were measured using digital calipers
(VWR, Brisbane, Calif.). The tumor volume was then calculated according
to the formula:

Volume=0.5236×length×width

which is based on an ellipsoidal shape, with the height of the tumor
estimated by the width, and has previously been used to approximate tumor
volume. Mice were euthanized when tumor volume exceeded 2 cm3.

[0187]Determination of Antibody Response in Blood by ELISA. As part of the
evaluation of the immune response to the administered vaccines, the IgG
antibody response to ovalbumin was measured for all mice in the tumor
study described in section 5.2.2. All reagents were obtained from Sigma
Aldrich Corp. (St. Louis, Mo.) unless otherwise noted. Blood was
collected by retro-orbital bleeding into heparin-coated capillary tubes
(VWR, Brisbane Calif.). The blood was transferred to 0.5 ml eppendorf
tubes and centrifuged for 10 min at 10,000 rpm. The plasma was then
collected and diluted 1:5000 in PBS. An Enzyme-Linked ImmunoSorbent Assay
(ELISA) was then performed to detect anti-ovalbumin IgG. First, 100 μl
of a 5 μg/ml ovalbumin solution was added to each well of a Nunc
Maxisorp 96-well plate and incubated overnight at 4° C. The plate
was aspirated and blocked with 150 μl of a 1% bovine serum albumin
(BSA) solution in PBS for 1.5 hrs at room temperature. The plate was then
washed 1× and 100 μl of each diluted mouse sample was added. All
samples were tested in triplicate. The samples were incubated for 3 hrs
at room temperature, then the plate was washed 3×. Goat anti-mouse
IgG (Fc Specific)-Peroxidase conjugate (Sigma product #A0168) was diluted
1:3000 in 0.1% BSA PBS-Tween and 100 μl was added to each well,
followed by a 2 hr incubation at room temperature. The plate was washed
3× and 100 μl of SureBlue Reserve TMB peroxidase substrate (KPL
Inc., Gaithersburg, Md.) was added. After 10 min, 100 μl 1N HCl was
added and the absorbance at 450 nm was recorded. In order to generate a
standard curve, mouse monoclonal anti-ovalbumin clone OVA-14 IgG1 (Sigma
product #A6075) was used at concentrations of 3 ng/ml to 200 ng/ml. All
washing steps were performed in PBS-Tween20 solution using a BioTek
(Winooski, VT) ELx50 plate washer.

[0188]Determination of CD8+Response in Splenocytes Using MHC-1 Tetramers.
In order to assess the ability of the PPAA carrier to provide CTL
activation, MHC-1 tetramers were used to evaluate the splenocytes of
immunized mice. Six-eight week old female C57B1/6 mice were obtained from
Jackson Labs. Mice (4 per group) were anesthetized with isofluorane and
immunized subcutaneously with 150 μl of PBS, ova, soluble PPAA-ova,
particulate PPAA-ova, soluble PMAA-ova, or particulate PMAA-ova, with an
equivalent of 100 μg ova delivered to each mouse. Seven days later
mice were euthanized and their spleens harvested. Spleens were
homogenized by forcing them through a 100 μm cell strainer into a
Petri dish containing DMEM culture medium with L-glutamine and sodium
pyruvate. Cells were counted and resuspended at 8×106
cells/ml. Phycoerythrin (PE)-conjugated iTAg® MHC class I tetramers
were purchased from Immunomics/Beckman Coulter (Fullerton, Calif.), and
the staining was carried out according to the provided protocol. 200
μl cell suspension was added to each flow cytometry tube. 5 μl
Mouse BD FcBlock (BD Biosciences, San Jose, Calif.) was added and
incubated for 5 min at 4C. Then, 10 μl of tetramer solution was added
in addition to 10 μl FITC rat anti-mouse CD8a antibodies (BD
Biosciences, San Jose, Calif.). Samples were vortexed gently and
incubated for 30 min at room temperature. Red blood cells were then lysed
by addition of 1 ml iTAg® MHC Tetramer Lyse Reagent and 25 μl
iTAg® MHC Tetramer Fixative Reagent. Samples were vortexed 5 seconds
and incubated for 10 min, then centrifuged at 150×g for 5 min. The
supernatant was removed, 3 ml PBS was added, and the centrifugation step
repeated. The cells were then resuspended in 500 μl PBS with 0.5%
paraformaldehyde and stored at 4° C. for 1 hour before analysis on
a FACScan 2 flow cytometer. Data was analyzed using FloJo software.

Example 5

Delivery of a Representative pH-Responsive Polymer Tumor Antigen Peptide
to Primary Dendritic Cells and Evaluation of Class I Presentation

PPAA-NY-ESO-1

[0189]Polymer-Peptide Conjugation. Peptides were obtained from EZBiolab
(Westfield, Ind.). PPAA-PDSA was synthesized as described in the
examples. Due to the hydrophobic nature of the peptide, the conjugation
reaction was performed in organic solvent. 3 mg PPAA-PDSA (Mw=17 kD,
Mn=6.2 kD, PDI=2.7) was dissolved in 150 μl dimethyl sulfoxide (DMSO).
0.53 mg of the Ny-Eso-1 peptide (MW 1.5 kD) peptide was then dissolved in
150 μl DMSO and added to the polymer solution. The reaction was
carried out at room temperature overnight. The absorbance at 372 nm
corresponding to released pyridine 2-thione was recorded to determine the
extent of reaction. The conjugate was dialyzed into slightly basic water
(MWCO 3.5 kD) for 1.5 days with frequent water changes, then lyophilized.

[0190]Evaluation of Class I Presentation in Human Primary Dendritic Cells.
Peripheral blood mononuclear cells (PBMCs) were collected from HLA-A2+
donors. Dendritic cells (DCs) were generated by exposing adherent cells
to 500 U/ml IL-4 and 800 U/ml GM-CSF in AIM-V culture medium (Invitrogen,
Carlsbad, Calif.). 1×105 DCs were then exposed to PPAA-peptide
conjugate or free peptide for 4 hours. DCs were washed and matured by
exposure to IL1β and LPS for 1 day, then incubated with
2×105 KJ-4 NY157-specific CTL clones. After 18 hrs, the
supernatant was collected and ELISA was performed to measure IFN-γ
produced by activated CTLs.

[0191]Use of Brefeldin A to Investigate Intracellular Antigen Processing
vs. External Peptide Loading. When DCs are pulsed with peptide, it is
possible for the peptide to externally load empty MHC molecules displayed
on the cell surface. In an attempt to distinguish between activation due
to antigens that have been internalized and processed vs. external
peptide loading, the ER transport and vesicle transport inhibitor
Brefeldin A (BFA) was used. The experiment was performed according to the
procedure described above with the addition of a group in which the DCs
were treated with BFA prior to exposure to the peptide/conjugate.

[0192]While illustrative embodiments have been illustrated and described,
it will be appreciated that various changes can be made therein without
departing from the spirit and scope of the invention.